Novel small molecule antimicrobials

ABSTRACT

Compounds and compositions of matter are provided that are small molecule antimicrobials. The compounds are selected from JH-144, TH-04 or TH-08 or combinations thereof. Compositions of matter are provided which in an embodiment may comprise a carrier, and in further embodiments may comprise a pharmaceutically acceptable excipient, film, biofilm or edible film. Methods of use are provided in which the composition may be administered to a subject in need thereof, and in an embodiment where the subject has a bacterial infection. Other embodiments provide the composition may be contacted with surface to eliminate or reduce bacteria.

REFERENCE TO RELATED APPLICATION

This application claims priority to previously filed and co-pendingprovisional application U.S. Ser. No. 62/361,846, filed Jul. 13, 2016,the contents of which are incorporated herein by reference in itsentirety and co-pending provisional application U.S. Ser. No. 62/306,986filed Mar. 11, 2016 the contents of which are incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present disclosure is related to broad-spectrum antimicrobialcompounds, compositions comprising the antimicrobial compounds, andmethods of treating/preventing bacterial infections with theantimicrobial compounds.

BACKGROUND OF THE INVENTION

Foodborne illnesses can result in major public health implications inthe U.S. and around the world. According to recently published CDC(Centers for Disease Control and Prevention) data foodborne diseasesaccount for approximately 8 million illnesses, and 9,000 deaths eachyear in the U.S. alone (CDC, 2011). The epidemiology of foodbomediseases is rapidly changing as newly recognized pathogens emerge andwell-studied pathogens increase in prevalence or associate with new foodvehicles. Apart from acute gastroenteritis, some foodbome diseases maycause chronic illness or disability. Listeriosis, for instance, cancause miscarriages or meningitis in patients with pre-existing chronicdiseases (Schuchat et al., 1991). As meat and meat products are themajor source of foodborne infection and the most important link betweenfood-producing animal and humans, the study of foodbome pathogensisolated from meat and poultry is indispensable.

Microbial contamination can reduce the shelf life of foods and increasethe risk of foodbome illness. This is a major worldwide public healthconcern. According to statistical data from the Center for DiseaseControl (CDC), five pathogen types account for over 90% of the estimatedfood-related deaths. Among these microorganisms, bacterial pathogens areSalmonella, Listeria, Campylobacter, E. coli 0157:H7 and Vibrio (CDC,2014) a significant causes of foodborne illnesses. Among these fivepathogens, illness associated with Campylobacter, termed“campylobacteriosis”, is one of the most common forms of bacterialfoodborne gastroenteritis in developed countries (Blaser et al., 2008).Apart from Campylobacter spp., the CDC estimates that E.coli 0157:H7causes approximately 73,000 illnesses and 60 deaths each year in theUnited States, in which 85% of these cases are attributed to foodbornetransmission. Listeriosis, another serious infection usually caused byeating foods contaminated with the bacterium Listeria monocytogenes, isalso an important public health problem. It is estimated that in theU.S., there are about 2500 cases with 500 deaths annually attributed tolisteriosis. The spectrum of listeriosis is broad, ranging fromasymptomatic infection and flu-like symptoms, to miscarriage,stillbirth, and meningitis (CDC, 2014).

Antimicrobial therapy is one of the most effective ways to prevent andcontrol bacterial diseases. Currently, the most commonly usedantimicrobials are macrolides (erythromycin) and fluoroquinolones(ciprofloxacin) with tetracycline used as an alternative (Moore et al.,2006). However, as the use of antimicrobials for therapy and prophylaxisincrease in both human and animal medicine, increasing numbers ofbacteria have developed resistance to these antimicrobials. As thesefoodborne pathogens are projected to remain top ten bacterial conditionsglobally, and several antibiotics are no longer effective in treatment ,a new generation of effective antimicrobials is critically needed.High-throughput, robust, cost-effective, phenotypic cell-based screeningis one such amenable approach to expedite antimicrobials discovery.Numerous studies (both laboratory and commercial plant-based)investigated potential interventions in processing plants to reduceCampylobacter on poultry carcasses. Evaluated measures includedfreezing, hot water treatment, irradiation, and chemicaldecontamination. Depending on the specific processing stage, severalpractices, such as time, temperature, pH, direction of water flow, andantimicrobial treatments can substantially affect the level of carcasscontamination by

Berrang, M. E., W. R. Windham, and R. J. Meinersmann. 2011.Campylobacter, Salmonella, and Escherichia coli on broiler carcassessubjected to a high pH scald and low pH postpick chlorine dip. Poult.Sci. 90:896-900.

An active food packaging system can serve as a non-thermal method toreduce foodborne pathogen contamination in food. By definition, activepackaging is defined as packaging in which subsidiary constituents havebeen deliberately included into the matrix or coated to the packagingmaterial, in the packaging headspace, to enhance the performance of thepackaging system (Robertson, 2006). One application of active packagingis to incorporate antimicrobial substances that can control microbialcontamination by reducing the growth rate and maximum population byextending the lag-phase of the target microorganism (Han, 2000). Theadvantage of this is that the antimicrobial agent does not have to beapplied directly to the food where it could be deactivated by additives(in the food), or by the processing methods, or by long term exposurebefore the food is ready for consumption. With active packaging, theantimicrobial agent can be made to contact the food at a strategic timeand it could be designed to be released into the product at a controlrate, thus its efficacy would be enhanced in terms of time andconcentration. The use of active antimicrobial packaging for the controlof microorganisms on food products is widely reported in the literature(Appendini, P., Hotchkiss, J. H. 2003, Review of antimicrobial foodpackaging. Innovative Food Science and Emerging Technologies. Vol. 3,113-126).

Five pathogens that account for over 90% of estimated food-relateddeaths: Samonella, Listeria, Campylobacter, E. coli O157:H7 and Vibrio(CDC, 2011can result in a self-limiting diarrheal illness in humans,however severe invasive diseases or prolonged illnesses inimmune-compromised individuals can occur and may require antimicrobialtherapy.

The genus Salmonella currently includes more than 2400 differentserotypes. Salmonella species are unique in the environment and cancolonize and cause disease in a variety of animals. Salmonellosis,caused by non-typhoidal Salmonella strains, typically results in aself-limiting diarrhea that do not need antimicrobial therapy, while insome rare cases, the infections of Salmonella enterica can lead tolife-threatening systematic syndrome which require effectivechemotherapy (Lee L. A., Puhr N. D., Maloney E. K., Bean N. H., Tauxe R.V., 1994, Increase in antimicrobial-resistant Salmonella infections inthe United States, 1989-1990, J. Infect. Dis. VOl. 170, pp.128-134). ForSalmonella spp., in some cases, a rapid spread through the animalproduction systems seems to have occurred at a global level in 1980s(Rodrigue, D. C., Tauxe, R. V., Rowe, B., 1990. International increasein Salmonella Enteritidis: a new pandemic? Epidemiology and Infection105, 21-27). It is reported that S .enteritidis appeared simultaneouslyaround most of European countries and U.S., and also spread into thepoultry production systems of developing countries later in the 1990s(Matope, G., Schlundt, J., Makaya, P. V., Aabo, S., Baggesen, D. L., 16J. Schlundt/International Journal of Food Microbiology 78 (2002) 3-171998. Salmonella Enteritidis in poultry: an emerging zoonosis inZimbabwe. Zimbabwe Veterinary Journal 29, 132-138).

Campylobacters are thin, curved, motile gram-negative rods. They aregenerally micro-aerophilic, though some strains are aerobic andanaerobic. Currently, campylobacters are recognized as the leading causeof foodborne gastroenteritis in the U.S. and one of the most frequentcauses of acute bacterial enteritis worldwide (Mead P. S., Slutsker L.,Dietz V., McCaig L. F., Bresee J. S., Shapiro C., 1999, Food-relatedillness and death in the United States, Emerg. Infect. Dis., pp.5607-5625). Gastroenteritis caused by Campylobacter is an acutediarrheal disease that typically causes high fever, abdominal cramping,and diarrhea that last from several days to more than one week. It is tobe noted that C. jejuni, and C. coli (clinically indistinguishable) arethe most common species associated with diarrheal illness, causing morethan 95% of Campylobacter enteritis (Harris N. V., Weiss N. S., Nolan C.M., 1986, The role of poultry and meats in the etiology of Campylobacterjejuni/coli enteritis, Am. J. Public Health Vol.76, pp. 407-411). Thereports of campylobacteriosis cases have been continuously increasing inmany parts of the world, as proved by statistical data from TexasDepartment of State Health Services. Most infections are sporadic singlecases resulting from the consumption of contaminated food, milk oruncooked and mishandled poultry (Friedman C. R., Neimann J., Wegener H.C., Tauxe R. V., 2000, In: I. Nachamkin, M. J. Blaser (Eds.),Campylobacter, 2nd ed., ASM Press, Washington D.C., pp. 130-130).

Shiga-toxin-producing Escherichia coli (STEC) was first recognized as anemerging human pathogen in 1982 when E. coli 0157:H7 was implicated intwo outbreaks of hemorrhagic colitis associated with consumption ofuncooked beef (Wells J. G., Davis B. R., Wachsmuth I. K., Riley L. W.,Remis R. S., Sokolow R., et al., 1983, Laboratory investigation ofhemorrhagic colitis outbreaks associated with a rare Escherichia coliserotype, J. Clin. Microbiol. Vol. 185, pp. 12-20). Human infection withSTEC can lead to non-bloody diarrhea or bloody diarrhea, or more seriousand fatal syndrome such as hemorrhagic colitis and hemolytic uremicsyndrome. It is proved that the most important virulence factorsassociated with STEC infection are Shiga toxins (stx1, stx2 or variants)(Schlundt J., 2002, New directions in foodborne disease prevention,International Journal of Food Microbiology Vol. 78, pp. 3-17).

Vibrio vulnificus is a gram-negative bacterium commonly found inestuarine and coastal habitats throughout the northern Gulf of Mexico.This species is an opportunistic human pathogen that can cause primarysepticemia, wound infection and gastroenteritis (Strom M. S., ParanjpyeR. N., 2000, Epidemiology and pathogenesis of Vibrio vulnificus.Microbes Infect. Vol 2, pp. 177-188). Comparing with gastroenteritis,primary septicemia is the most common and severe syndrome caused by V.vulnificus, with mortality rate of more than 50% (Blake P. A., 1979,Disease caused by a marine vibrio—clinical characteristics andepidemiology. N. Engl. J. Med. Vol. 300, pp. 1-5). Most reported casesrevealed that consumption of raw shellfish and eastern oyster is themain cause of infections (Strom et al., 2000). Besides, V. vulnificuscan produce severe skin and soft tissue infections in patients withpre-existing wounds who come in contact with the bacterium via seawateror by handling seafood (Howard R. J., Lieb S., 1988, Soft-tissueinfections caused by halophilic marine vibrios. Arch. Surg. Vol.123, pp.245-249).

Many studies indicated that Listeria monocytogenes grows well atrefrigeration temperatures and with minimal nutrients, and is able tosurvive and even grow in plants, soil and water (Schlundt J., 2002, Newdirections in foodborne disease prevention, International

Journal of Food Microbiology Vol. 78, pp. 3-17). The foodbornetransmission has been recognized as a major source of human listeriosissince 1982, though the first reported human listeriosis was in 1929. Thewidespread nature of L. monocytogenes allows easy access to foodproducts during various phases of production, processing, manufacturing,and distribution, thus it has been found in many food products,including fresh vegetables, raw milk, raw meats, and eggs. Manyillnesses are associated with refrigerated processed foods(ready-to-eat) consumed without prior cooking or reheating. Theincidence of listeriosis has increased over the past two decadesthroughout the world. It is estimated by CDC that in the U.S., there are2500 cases with 500 deaths attributed to listeriosis annually, mostlyinvolving pregnant women, newborn babies, the elderly, andimmune-compromised people. The spectrum of listeriosis is broad, rangingfrom asymptomatic infection and flu-like symptoms, to miscarriage,stillbirth, and meningitis (Robert R., 2003).

The prevention and control of foodborne disease depends on GoodManufacturing Practices (GMP) of food production, including the handlingof raw ingredients and the preparation of finished products. If not,hazards can be introduced at any point from the farm to the table. Theintroduction of the Hazard Analysis Critical Control Point (HACCP)system greatly improved hygiene control in processing plants. Suchprograms require food industries to identify points in food productionwhere contamination may occur and target resources toward processes thatreduce or eliminate foodborne hazards (Goodfellow, S. J. 1995.Implementation of the HACCP program by meat and poultry slaughterers. InHACCP in Meat, Poultry and Fish Processing, eds. A. M. Pearson & T. R.Dutson, pp. 58-71. Glasgow, UK, Blackie Academic & Professional).However, infections cannot be fully eliminated since young animals orpoultry are highly susceptible to pathogens. Milner and Shafferindicated that the infective dose of S. typhimurium for one-day-oldbirds with an oral administration was as low as 10 CFUs (Milner K. C.,Shaffer, M. F. 1952. Bacteriologic studies of experimental Salmonellainfections in chicks. Journal of Infectious Diseases Vol. 90, pp.81-96). This deficiency could be overcome by oral administration of asaline suspension with mature micro-flora from adult birds. In this way,adult-type microflora would be established in young birds and thusprevent them from pathogenic infections by the phenomenon known as“competitive exclusion” (Rantala, M., Nurmi, E. 1973. Prevention of thegrowth of Salmonella infantis in chicks by the flora of the alimentarytract of chickens. British Poultry Science Vol. 14, pp. 627-30).

As can be seen, there is a continuing need to develop newantimicrobials. Other objects, aspects and advantages of this inventionwill be apparent to one skilled in the art in view of the followingdisclosure, the drawings, and the appended claims.

SUMMARY OF THE INVENTION

Applicants have synthesized new antimicrobial compounds JA-144, TH-04,or TH-08 which form the basis of this application. The inventionincludes these compounds as well as derivatives, modifications, orpharmaceutically acceptable salts thereof. In another embodimentincluded herein is an antimicrobial compound having the formula forJA-144, TH-08, or TH-04 as set forth below:

Also included are antimicrobial compositions including theaforementioned compounds and a carrier, preferably a film carrier, morepreferably an edible film. Yet a further embodiment includes methods oftreating a subject, surface or substrate in need of treatment for abacterial contamination or infection. Subjects or surfaces are thencontacted with one or more the compositions of the invention to reduce,prevent or treat bacterial infection. In still further embodiments theantimicrobial compounds are impregnated within a film coating to beapplied to a substrate or food substance. An embodiment provides thefilm may be a packaging material in contact with a surface or foodsubstance. In yet another embodiment the substrate or surface is a foodsurface or substrate and the film is an edible film coating.

In still another embodiment, the invention includes methods of reducingthe antimicrobial activity of Gram-negative bacteria, Gram-positivebacteria, or bacteria that are neither Gram-positive nor Gram-negative.In a still further embodiment the Gram-negative bacteria is Escherichiacoli, Pseudomonas aeruginosa, Candidatus liberibacter, Agrobacteriumtumefaciens, Branhamella catarrhalis, Citrobacter diversus, Enterobacteraerogenes, Klebsiella pneumoniae, Proteus mirabilis, Salmonellatyphimurium, Neisseria meningitidis, Serratia marcescens, Shigellasonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii,Salmonella enteriditis, Fusobacterium nucleatum, Veillonella parvula,Bacteroides forsythus, Actinobacillus actinomycetemcomitans,Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis,Helicobacter pylori, Francisella tularensis, Yersinia pestis, Vibriocholera, Shigella boydii, Morganella morganii, Edwardsiella tarda,Campylobacter jejuni, Campylobacter coli or Haemophilus influenzae. Inanother embodiment the Gram-positive bacteria is a species of Bacillus,Listeria, Staphylococcus, Streptococcus, Enterococcus, Corynebacterium,Propionibacterium or Clostridium. In yet another embodiment theGram-positive bacteria is Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcuspyogenes, Bacillus cereus, or Bacillus anthraces.

BRIEF SUMMARY OF THE DRAWINGS

FIG. 1 is a graph showing the formula of compounds JA-144, TH-04, TH-08and JA-128.

FIG. 2 are graphs showing Primary HTS of compounds for growth inhibitionagainst C. jejuni 81-176 using a pre-selected library of 1,182compounds. A cut off percentage value of ≥99.0% growth inhibitionresulted in 781 hit compounds. These compounds were categorized based ontheir activity (see details in box labeled B).

FIG. 3 is a graphic showing synthesized analogues of compound 1.

FIG. 4 are graphs showing in vivo effect of TH-4 and TH-8 on C. jejunicolonization in three week old broiler chickens. JA-144, anotherresynthesized compound had broad spectrum effect on other bacteriaincluding E. coli and Listeria.

FIG. 5 is a graphic showing the process of synthesis of compound 1.

FIGS. 6A and B are graphics showing BarT sequence collections (A) andstats (B), here an example of Yeast.

FIG. 7 is a graph showing percent growth inhibition against C. jejuniwith 15 compounds.

FIG. 8 is a graph showing growth inhibition against C. coli of 15compounds.

FIGS. 9A and B are graphs showing TH-4 effect (A) on C. jejuni growthand C. coli growth (B).

FIGS. 10A and B are graphs showing TH-8 effect on C. jejuni growth (A)and C. coli growth (B).

FIG. 11 is a graph showing efficacy of TH-7 and TH-8 compounds whenincorporated in edible films against C. jejuni and C. coli.

FIG. 12 is a graph showing C. jejuni growth inhibition by JA-144 andJA-128.

FIG. 13 are graphs showing JA-144 effect on C. jejuni growth (A) and C.coli growth (B).

FIG. 14 is a graph showing efficacy of JA-144 when incorporated inedible films against four bacteria.

FIG. 15 is a graph showing dose-dependent cytotoxicity of compounds onnormal colon cells.

FIG. 16 is a graph showing dose-dependent cytotoxicity of JA-144 andJA-128 and Paclitaxel.

FIG. 17 is a graph showing E. coli growth curve when exposed to JA-144at varying concentrations.

FIG. 18 is a graph showing L. innocuous growth curve when exposed toJA-144 at varying concentrations.

FIG. 19 is a graph showing C. jejuni growth curve when exposed to JA-144at varying concentrations.

FIG. 20 is a graph showing C. jejuni growth curve when exposed to TH-4at varying concentrations.

FIG. 21 is a graph showing C. jejuni growth curve when exposed to TH-8at varying concentrations.

FIGS. 22A and B are graphs showing log reduction of bacteria whenexposed to JA-144 in broth (A) and in tapioca film (B).

FIGS. 23A and B are graphs showing log reduction of C. jejuni (left bar)when exposed to JA-144, TH-4 and TH-8 in broth and control (rightbar)(A) and C. coli (B).

FIGS. 24A and B are graphs showing log reduction of C. jejuni (left bar)when exposed to JA-144, TH-4 and TH-8 in broth and control (right bar)(A) and C. coli (B).

FIGS. 25A and B are graphs showing moisture content (A) and wateractivity (B) of tapioca films with JA-144, TH-4 or TH-8.

FIG. 26 is a graph showing water vapor permeability for tapioca filmswith JA-144, TH-4 or TH-8.

FIG. 27 is a graph showing oxygen permeability coefficient for tapiocastarch with JA-144, TH-4 or TH-8.

FIG. 28 is a graph showing differential scanning calorimetry of tapiocafilms with JA-144, TH-4 or TH-8 at 1%

FIGS. 29A-D are graphs showing X-ray diffraction of tapioca films withJA-144, TH-4 or TH-8.

FIG. 30 is a graph showing the effect of JA-144, TH-4 or TH-8 on thestorage modulus of tapioca films.

FIG. 31 is a graph showing loss modulus of tapioca films with JA-144,TH-4 or TH-8.

FIG. 32 is a graph showing Tan Delta of tapioca films with JA-144, TH-4or TH-8.

FIG. 33 is a graphic showing possible mechanism of JA-144 binding toamylose or glycerol.

DETAILED DESCRIPTION OF THE INVENTION

Described here are antimicrobial compounds JA-144, TH-04, or TH-08(sometimes here referred to as TH-4 or TH-8) and which includesderivatives, modifications, or pharmaceutically acceptable saltsthereof.

Examples of these are provided in the Examples below. Derivatives of thecompounds include, but are not limited to, any salt, ester, acids,bases, solvates, hydates, and prodrugs. Derivatives, modifications andpharmaceutically acceptable salts retain the functional propertiesdescribed herein.

“Pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds wherein the parent compound is modified by making anacid or base salt thereof, and further refers to pharmaceuticallyacceptable solvates of such compounds and such salts. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalsalts and the quaternary ammonium salts of the parent compound formed,for example, from inorganic or organic acids. For example, conventionalacid salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC—CH₂)_(n)—COOH where n is 0-4, andthe like. The pharmaceutically acceptable salts of can be synthesizedfrom a parent compound that contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate, or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, non-aqueous media like ether,ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred,where practicable.

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a group atleast one group selected from a halide (F—, Cl—, Br—, I-), a hydroxyl, aC₁ to C₂₀ alkoxy, a cyano, a C₁ to C₂₀ alkyl, a C₂ to C₁₆ alkenyl, a C₂to C₁₆ alkynyl, a C₆ to C₂₀ aryl, a C₇ to C₁₃ arylalkyl, a C₇ to C₁₃aryloxyalkyl, a C₇ to C₁₃ arylthioalkyl, a C₁ to C₂₀ heteroalkyl, a C₃to C₂₀ cycloalkyl, and a c₅ to C₁₅ heterocycloalkyl, provided that thedesignated atom's normal valence is not exceeded. When a substituent isoxo (i.e., ═O), then 2 hydrogens on the carbon atom are replaced. Whenaromatic moieties are substituted by an oxo group, the aromatic ring isreplaced by the corresponding partially unsaturated ring. For example apyridyl group substituted by oxo is a pyridone. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds or useful synthetic intermediates. A stablecompound or stable structure is meant to imply a compound that issufficiently robust to survive isolation from a reaction mixture, andsubsequent formulation into an effective therapeutic agent.

Applicants have synthesized novel compounds, from those identified froma screen of a small molecule library of 4, 182 compounds forbactericidal activity against C. jejuni. 781 compounds were identifiedpossessing bactericidal or bacteriostatic activity. Through secondaryscreens applicants identified several compounds with narrow spectrumactivity specific to C. jejuni and C. Coli. From these, Applicants thensynthesized 15 derivatives of one of the compounds and from these, threesuperior novel derivatives were identified.

In one aspect, provided herein are methods of treating an animal subjectin need of treatment for a bacterial infection, comprising administeringto the individual an antimicrobial compound or composition as describedherein. The bacteria can be actively growing or in the stationary phase.In one aspect, administration of an antimicrobial compound is topicaladministration. In another aspect, administration of an antimicrobialcompound is systemic administration such as oral administration. Inother embodiments surfaces or substrates may be treated with thecompounds of the invention for reduction or inhibition of bacterialcontamination such as food surfaces, packaging, or equipment, primarilyrelated to meat and poultry products or production.

The bacteria causing the infection can be Gram-negative, Gram-positive,or bacteria that are neither Gram-negative nor Gram-positive.Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa,Candidatus liberibacter, Agrobacterium tumefaciens, Branhamellacatarrhalis, Citrobacter diversus, Enterobacter aerogenes, Klebsiellapneumoniae, Proteus mirabilis, Salmonella typhimurium, Neisseriameningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii,Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteriditis,Fusobacterium nucleatum, Veillonella parvula, Bacteroides forsythus,Actinobacillus actinomycetemcomitans, Aggregatibacteractinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori,Francisella tularensis, Yersinia pestis, Vibrio cholera, Shigellaboydii, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni,Campylobacter coli and Haemophilus influenzae. In another embodiment,the bacteria are Gram-positive bacteria. Gram-positive bacteria includespecies of Bacillus, Listeria, Staphylococcus, Streptococcus,Enterococcus, Corynebacterium, Propionibacterium and Clostridium.Specific Gram-positive bacteria include Staphylococcus aureus,Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis,Streptococcus pyogenes, Bacillus anthraces and Bacillus cereus. In aspecific embodiment, the bacteria are one or more drug resistantbacteria. Bacteria that are neither Gram-negative nor Gram-positiveinclude Borrelia burgdorferi, Mycobacterium leprae, Mycobacteriumtuberculosis and other Mycobacteria. Further included are bacteria suchas Chlamydia and Mycoplasma that do not have a cell wall. In certainaspects, the bacteria are resistant bacteria such ascarbapenam-resistant bacteria, methicillin-resistant Staphylococcusaureus, vanccomycin-resistant Enterococci or multi-drug resistantNeisseria gonorrhoeae.

In another aspect, a method of inhibiting bacterial growth comprisescontacting the bacteria with an antimicrobial compound as describedherein. The bacteria can be actively growing or in the stationary phase.Methods of inhibiting bacteria include methods useful for treatment of asubject (human or veterinary) and also include methods useful forinhibiting bacteria outside of a subject, such as use for sterilizationand disinfection.

In one embodiment, the bacteria are in the form of a biofilm. A biofilmis a complex aggregate of microorganisms such as bacteria, wherein thecells adhere to each other on a surface. The cells in biofilms arephysiologically distinct from planktonic cells of the same organism,which are single cells that can float or swim in liquid medium. Biofilmsare involved in, for example, urinary tract infections, middle earinfections, dental plaques, gingivitis, coatings of contact lenses,cystic fibrosis, and infections of joint prostheses and heart valves.

The antimicrobial compounds and compositions may be administeredprophylactically, chronically, or acutely. For example, such compoundsmay be administered prophylactically to animal subjects known to beprone to bacterial infections, or who are known to have been exposed topotentially infectious agents.

Since the antimicrobial compounds are antibacterially active and inhibitbacterial growth, they are also of use in treating bacterialcontamination of a substrate, such as hospital instruments or worksurfaces. In order to treat a contaminated substrate, the compounds maybe applied to the site of such contamination in an amount sufficient toinhibit bacterial growth.

In certain embodiments, the compounds are administered to an animalsubject. A “subject”, used equivalently herein, means mammals andnon-mammals. “Mammals” means a member of the class Mammalia including,but not limited to, humans, non-human primates such as chimpanzees andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, and swine; domestic animals such as rabbits, dogs, andcats; laboratory animals including rodents, such as rats, mice, andguinea pigs; and the like. Examples of non-mammals include, but are notlimited to, birds, and the like. The term “subject” does not denote aparticular age or sex.

The phrase “effective amount,” as used herein, means an amount of anagent, which is sufficient enough to significantly and positively modifysymptoms and/or conditions to be treated (e.g., provide a positiveclinical response). The effective amount of an active ingredient for usein a pharmaceutical composition will vary with the particular conditionbeing treated, the severity of the condition, the duration of thetreatment, the nature of concurrent therapy, the particular activeingredient(s) being employed, the particular pharmaceutically-acceptableexcipient(s)/carrier(s) utilized, and like factors within the knowledgeand expertise of the attending physician. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art.

The phrase “inhibitory amount”, as used herein, means an amount of anagent (a compound or composition), which is sufficient to reduce thelevel or activity of bacterial infection to a statistically significantlesser value as compared to when the agent is not present.

The amount of compound effective for any indicated condition will, ofcourse, vary with the individual subject being treated and is ultimatelyat the discretion of the medical or veterinary practitioner. The factorsto be considered include the condition being treated, the route ofadministration, the nature of the formulation, the subject's bodyweight, surface area, age and general condition, and the particularcompound to be administered. In general, a suitable effective dose is inthe range of about 0.1 to about 500 mg/kg body weight per day,preferably in the range of about 5 to about 350 mg/kg per day. The totaldaily dose may be given as a single dose, multiple doses, e. g., two tosix times per day, or by intravenous infusion for a selected duration.Dosages above or below the range cited above may be administered to theindividual patient if desired and necessary.

The compounds may be combined in an embodiment with a carrier, which canbe a pharmaceutically acceptable excipient and/or diluent appropriatefor the process in which it will be used. Where administered to ananimal, it will be non-toxic to the animal. The carrier, excipientand/or diluent is provided to provide improved properties of thecomposition, such as standardizing, preserving and stabilizing, allowingthe bacteria or component to survived the digestive system of an animal,lubrication, and improve delivery. There are a myriad of such agentsavailable which may be added. Without intending to be limiting, examplesinclude wetting agents and lubricating agents, preservative agents,lipids, stabilizers, solubilizers and emulsifiers

Also included herein are pharmaceutical compositions comprising theantimicrobial compounds. As used herein, “pharmaceutical composition”means a therapeutically effective amount of the compound together with apharmaceutically acceptable excipient, such as a diluent, preservative,solubilizer, emulsifier, adjuvant, and the like. As used herein“pharmaceutically acceptable excipients” are well known to those skilledin the art. In one aspect, a pharmaceutical composition is suitable fortopical administration. In another aspect, a pharmaceutical compositionis suitable for systemic administration.

Tablets and capsules for oral administration may be in unit dose form,and may contain excipients such as binding agents, for example syrup,acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillersfor example lactose, sugar, maize-starch, calcium phosphate, sorbitol orglycine; tableting lubricant, for example magnesium stearate, talc,polyethylene glycol or silica; disintegrants for example potato starch,or acceptable wetting agents such as sodium lauryl sulfate. The tabletsmay be coated according to methods well known in normal pharmaceuticalpractice. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations that may beused for the drug are conventional formulations well known in the art.Topical administration includes transdermal formulations such aspatches.

The active ingredient may also be administered parenterally in a sterilemedium, either subcutaneously, or intravenously, or intramuscularly, orintrasternally, or by infusion techniques, in the form of sterileinjectable aqueous or oleaginous suspensions. Depending on the vehicleand concentration used, the drug can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agents can be dissolved in the vehicle.

Pharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage” or “unit dose” means a predeterminedamount of the active ingredient sufficient to be effective for treatingan indicated activity or condition. Making each type of pharmaceuticalcomposition includes the step of bringing the active compound intoassociation with a carrier and one or more optional accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidor solid carrier and then, if necessary, shaping the product into thedesired unit dosage form.

The antimicrobial compounds may also be administered in combination withan additional active agent, such as, for example, an inhibitor ofbacterial efflux. Efflux pumps are proteins that unidirectionally removeantibiotics from cytoplasmic compartments, and are considered to be amechanism of antibacterial resistance. Bacterial efflux inhibitorsinclude chalcone compounds as disclosed in WO 11/075136, the polybasiccompounds disclosed in WO 10/054102, the quaternary alkyl ammoniumfunctional compounds disclosed in WO 08/141012, the compounds disclosedin WO 05/007162, the substituted polyamines of WO 04/062674, which areincorporated herein by reference in their entirety.

In another embodiment, the antimicrobial compounds of Formula I can beadministered with a second antibiotic. Exemplary second antibioticsinclude, for example, glycopeptides (e.g, vancomycin or teicoplanin);penicillins, such as amdinocillin, ampicillin, amoxicillin, azlocillin,bacampicillin, benzathine penicillin G, carbenicillin, cloxacillin,cyclacillin, dicloxacillin, methicillin, mezlocillin, nafcillin,oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin;cephalosporins, such as cefadroxil, cefazolin, cephalexin, cephalothin,cephapirin, cephradine, cefaclor, cefamandole, cefonicid, ceforanide,cefoxitin, and cefuroxime, cefoperazone, cefotaxime, cefotetan,ceftazidime, ceftizoxime, ceftriaxone, and moxalactam; carbapenems suchas imipenem; monobactams such as aztreonam; tetracyclines such asdemeclocycline, tigilcycline, doxycycline, methacycline, minocycline,and oxytetracycline; aminoglycosides such as amikacin, gentamicin,kanamycin, neomycin, netilmicin, paromomycin, spectinomycin,streptomycin, and tobramycin; polymyxins such as colistin,colistimathate, and polymyxin B, and erythromycins and lincomycins andalso sulfonamides such as sulfacytine, sulfadiazine, sulfisoxazole,sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim,quinolones, novobiocin, pyrimethamine, rifampin, quinolines,fluoroquinolines; and combinations thereof.

In another embodiment the compounds of the invention may be appliedthrough a film, which in certain embodiments may be a biofilm or ediblefilm. A film includes coatings or layers of compositions placed adjacenta surface. An edible film is a thin layer acting as a barrier betweenfood and its surrounding environment. This layer can also be consumedwith the food since it is edible. These coatings and films are designedto prolong the quality and shelf life of food by protecting it fromphysical, mechanical or biological damage (Janjarasskul T., Krochta J.M. 2010, Edible Packaging materials. Annu Rev Food Sci Technol. Vol. 1,pp. 415-448). Such films are well known to one skilled in the art andoptions and methods of producing such films will vary depending upon theparticular application (see, for example, Nitin et al WO2015084938;Krochta et al U.S. Pat. No. 5,543,164). An example of an edible film isthe sausage casing, which is not removed during cooking or eating.General functions of edible coatings and films in food processing arefor: enhancing quarantined treatments, improving the appearance of thefood, incorporation of flavors and pigments, reduction in loss offlavors and aromas, reduction in gas diffusion and reduction in waterloss (Krochta, J. M., 2002. Proteins as raw materials for films andcoatings: Definitions, current status and opportunities in Protein-BasedFilms and Coatings. Boca Raton, Fla.: CRC Press, pp. 672).

Edible films and coatings can either be prepared from lipids,polysaccharides or proteins, or from a combination of them (Dangaran, K.L. and J. M. Krochta. 2008. Whey protein films and coatings. In WheyProcessing, Functionality and Health Benefits. C. Onwulata and P. Huth(eds.) Blackwell Publishing, Ames, Iowa). Some of the polysaccharidesthat are suitable for use as edible films and coatings include chitosan,starches, pectin, alginates and cellulose derivatives (Mohammed A. 2010,Chitosam application for active bio-based films production and potentialin the food industry: Review. Food Science and Technology, Vol. 43,Issue. 6, pp. 837-842). Typically they have good oxygen but poormoisture barrier properties due to their hydrophilic nature and theability to form strong hydrogen bonding that can be used to cross-linkwith functional additives such as flavors, colors, and micronutrients.Animal and vegetable fats used to make films and coatings are compoundssuch as fatty acids,acylglycerols, and waxes. These lipid compounds arequite suitable because they are excellent barriers to moisture and theyadd extra gloss to confectionary products. Waxes are mainly utilized ascoatings on fruits and vegetables to reduce loss of moisture and retardrespiration (Valérie M., Frédéric D., Geneviève B., Martine C., AndréeV., 2010, Factors Affecting the Moisture Permeability of Lipid-BasedEdible Films: A Review. Critical Reviews in Food Science and Nutrition.Vol. 42, Issue. 1, pp.67-89). Apart from the preservation factor, thesefilms and coatings also facilitate the incorporation of food additivesinto the food to enhance the texture, flavor and color (Cagri A.,Ustunol, Z., Ryser, E., 2004. Antimicrobial edible films and coatings.Journal of Food Protection Vol. 67. pp.833-848). Protein based films arealso hydrophilic and have good mechanical strength and can be used forthe individual packaging of small portions of food, such as beans, nutsand cashew nuts. Moreover, they serve as functional carriers forantimicrobial and antioxidant agents (Thawien W., 2012, Protein-BasedEdible Films: Characteristics and Improvement of Properties.Agricultural and Biological Sciences, “Structure and Function of FoodEngineering, Chapter 3.” ISBN: 978-953-51-0695-1).

The use of edible coatings and films continues to expand due to theirjustification by research findings in the field of active packaging. Oneof these active packaging applications is the incorporation ofantimicrobial agents into or on edible films that can be used tosuccessfully inhibit spoilage and pathogenic organisms from infiltratingfood products. Examples of antimicrobial agents include essential oilsand plants extracts, enzymes, chitosan, bacteriocins, nanoparticles andsmall molecule drugs (Suet-Yen S., Lee T. S., Tiam-Ting T., Soo-TueenB., Rahmat A R., Rahman W. A. W. A., Ann-Chen T., Vikhraman M., 2013,Antimicrobial agents for food packaging applications. Trends in FoodScience & Technology Vol. 33, pp. 110-123). However, there are stillsome challenges in the application of films and coatings. For example,environmental factors such as temperature and humidity that cannot becompletely controlled during transportation and storage tend to makehydrophilic films more permeable (Pascall M. A., Lin S. J., 2013. TheApplication of Edible Polymeric Films and Coating in the Food Industry.Journal of Food Processing & Technology. Vol. 71, pp. 95-101).

In a preferred embodiment the invention includes an edible filmincorporated with small molecules to inhibit the growth of Campylobacterjejuni and Campylobacter coli in raw chicken & poultry products. Anideal film should have some characteristics such as good oxygen barrierproperty to prevent the growth of aerobic bacteria, good MIC value ofsmall molecules when incorporated in the film, good mechanical propertyto wrap the poultry products as well as non-toxic, unflavored andcolorless.

Types of Edible Film Material

Generally the main components of daily-consumed foods, such as proteins,carbohydrates and lipids can meet the requirements for the preparationof edible films. As a rule of thumb, lipids are used to reduce watertransmission, since lipids have good moisture barrier. However, theyhave low mechanical properties due to their hydrophobic structure andtheir inability to form cross linkages. Polysaccharides are used tocontrol oxygen and other gas transmission, because they are hydrophilicand provide strong hydrogen bonding that can be used to cross-link withfunctional additives such as flavors, colors, and micronutrients.Protein films, on the other hand, have good mechanical stability so thatwhen applied to fruits, they help to reduce injuries duringtransportation. These materials can be utilized individually or as mixedcomposite blends to form films. The main types of edible films includealginate, carrageenan, cellulose and it derivatives, dextrin, pectin andstarch are examples of polysaccharide films. Protein films can be madefrom various sources, such as corn, milk, soy, wheat and whey. Lipidbased films such as waxes, glycerol esters, and resins are the oldestknown edible film components, however, they are less widely usedcurrently due to their susceptibility to oxidation and low mechanicalstrength.

Protein-Based Edible Film/Coating.

Protein-based edible films have triggered interest in recent yearsbecause of their advantages, including the use of edible packagingmaterials for individual packaging of small portions of food, such asbeans and nuts. In addition, they can be applied between heterogeneousfood at the interfaces between different layers of components to preventthe transfer of inter-component moisture and solute migration in pizzas,pies and candies, for example (Bryne Mubururu, Dinga N. Moyo, PerkinsMuredzi. Production of Artificial Sausage Casings from Whey Proteins.International Journal of Nutrition and Food Sciences. Special Issue:Optimizing Quality and Food Process Assessment. Vol. 3, No. 6-1, 2014,pp. 30-38). Besides, they can also function as carriers forantimicrobial and antioxidant agents.

In its natural state, protein films can be divided into two groups,fibrous protein films and globular protein films (Thawien, 2012).Fibrous protein chains are water insoluble, fully extended, andassociated closely with each other in parallel structures via hydrogenbonding to form fibers. Globular proteins are soluble in water or acidbased solutions. They fold into complicated spherical structures heldtogether by a combination of hydrogen, ionic, hydrophobic and covalentbonds. With regard to fibrous proteins, collagen has received the mostattention as a protein-based film, while for globular proteins; examplesare corn zein and whey protein.

Collagen

Collagen is the main protein of connective tissue such as bone, hide,tendons cartilage and ligaments. Due to its biological properties andready availability, which is unique among those of natural polymers,type I collagen is widely used as a biomaterial (Sisken B. F, Zwick M.,Hyde J. F., Cotrill C. M., 1993, Maturation of the central nervoussystem: comparison of equine and other species. Equine Veterinary, Vol.25, Issue. 14, pp. 2042-3306). It is the most commercially successfuledible protein film due to its biocompatible and non-toxiccharacteristics, and its structural, physical, chemical andimmunological properties. It can be produced into a variety of forms,and can be easily isolated and purified in large quantities (Hood E. E.,Shen Q. X., Varner J. E., 1988, A developmentally regulatedhydroxyproline-rich glycoprotein in maize pericarp cell walls. PlantPhysiology, Vol. 1, pp. 138-142).

Corn Zein

Corn Zein is the major protein in corn. Due to its high content ofnon-polar amino acids, it is hydrophobic and is thermoplastic in nature(Shukla R., Cheryan M., 2001, Zein: The industrial protein from corn.Industrial Crops and Products, Vol. 13, Issue. 3, pp. 171-192). Cornzein has excellent film-forming properties and can be used for thefabrication of biodegradable films. The formation of corn zein films isfacilitated by the development of hydrophobic, hydrogen and limiteddisulfide bonds between zein chains in the film matrix (Gennadios A.,Hanna M. A., Kurth L. B., 1997, Applications of edible coatings onmeats, poultry and seafoods: a review. Food Science and Technology, Vol.30, Issue. 4, pp. 337-350), this however, results in the formation ofbrittle films that require the addition of plasticizers to enhance itsflexibility. Its hydrophobicity characteristic enables good water vaporbarrier property when compared to other edible films. It also shows theability to reduce moisture and loss of firmness and delay color changein fresh fruit (Guilbert S., Gontard N., Cup B., 1986, Technology andapplication of edible protective films. Packaging Technology andScience. Vol. 8, Issue. 6, pp. 339-346).

Whey Protein

Whey protein is a nutritional and highly functional protein. It isformed through the use of transglutaminase as a crosslinking agent(Mahmoud, R., Savello, P. A. 1993, Solubility and hydrolyzability offilms produced by transglutaminase catalytic crosslinking of wheyprotein. J. Dairy Sci. Vol. 76, Issue. 1, pp. 2935), and is shown toproduce a transparent, bland, flexible, water-based edible film withexcellent oxygen, aroma and oil barrier properties at low relativehumidity (Miller K. S., Krochta J. M., 1997, Oxygen and aroma barrierproperties of edible films: A review. Trends in Food Science & Tech.Vol. 8, Issue. 7, pp. 228-237). The most beneficial characteristic ofwhey protein edible films is their edibility and inherentbiodegradability (Krochta, J. M., 2002. Proteins as raw materials forfilms and coatings: Definitions, current status and opportunities inProtein-Based Films and Coatings. Boca Raton, Fla.: CRC Press, pp. 672),especially the latter feature, which is attractive to the food industrybecause it reduces the bio-burden on the environment. However, to be ofuse in food packaging, the shelf life of the edible film should belonger than the shelf life of the packaged product (Krochta, J. M., DeMulder-Johnston, C. D., 1997, Edible and biodegradable polymer films:Challenges and opportunities. Food Technol, Vol. 51, pp. 61-74).

Polysaccharide-Based Edible Film/Coating

Polysaccharide films are made from starch, alginate, cellulose ethers,chitosan, carrageenan, or pectin. The great diversity of structuralcharacteristics of polysaccharides is exhibited differences in itsmonosaccharide composition, linkage types and patterns, chain shapes anddegree of polymerization, which influences hardness, crispiness,compactness, thickening quality, viscosity, adhesiveness, and gelforming ability. Typically, polysaccharide-based edible films haveexcellent gas permeability properties due to the hydrogen bondsformation between two hydrophilic subunits, thus enhancing the shelflife of the product without creating anaerobic conditions (Baldwin etal., 1995). They could also be used to extend the shelf life of musclefoods by preventing dehydration, oxidative rancidity, and surfacebrowning. However, the hydrogen-bonding characteristic makes them poorbarriers for water vapor.

Starch

Starch, is composed of amylose and amylopectin and is primarily derivedfrom cereal grains such as corn, wheat, potato tapioca, and rice. Starchis typically found as granules, which contain millions of amylopectinmolecules accompanied by even larger numbers of smaller amylosemolecules (Whistler R. L., Daniel J. R., 1985. Carbohydrates. In O. R.Fennema (Ed.), Food chemistry (2nd ed.) pp. 69-137). New York: MarcelDekker. Amylose is responsible for the film forming capacity of starch(Claudia A. R. B., Bello-Perez L. A., Gacia M. A., Martino M. N.,Solorza-Feria J., Zaritzky N. E., 2005, Carbohyd. Polym. Vol. 3, Issue.2, pp. 156-161). Films with high amylose content are flexible; show lowoxygen permeability, heat-sealable, oil resistant, but water-soluble.Starch-based films are odorless, tasteless, colorless, non-toxic,biologically absorbable, and resistant to the passage of oxygen (KrogarsK., Heinamaki J., Karjalainen M., Rantanen J., Luukkonen P., YliruusiJ., Eur J., 2003, Development and characterization of aqueousamylose-rich maize starch dispersion for film formation. Pharm.Biopharm. Vol. 56, Issue. 2, pp. 215-221).

Carrageenan

Carrageenan is a water-soluble polymer with a linear chain of partiallysulfated galactans, which plays a role in its film-forming ability. Itis extracted from the cell walls of various red seaweeds (Rhodophyceae).Variations in the degree of sulfate groups present in its structuredivide carrageenan into three types: kappa, iota, and lambdacarrageenan.

The presence of hydroxyl and sulfate groups in the structure ofcarrageenan causes its hydrophilic nature. It is widely used as an agentfor thickening and gelling in food and nonfood industries due to itswater holding ability (Van de Velde F.,Lourenço, N. D., Pinheiro H. M.,Bakker M., 2002, Carrageenan: A food-grade and biocompatible support forimmobilization techniques. Adv Synth Catal, No. 344, pp. 815-835).Besides, kappa carageenans have the ability to form thermoreversiblegels. However, in the presence of a small amount of acid, thischaracteristic will be disrupted due to cross-link formation as a resultof the presence of extra positive ions (Park et al., 2001).

Cellulose Derivatives

Cellulose derivatives are polysaccharides composed of linear chains ofbeta-1,4 glucosidic units with methyl, hydroxypropyl or carboxylsubstituents. Generally, there are four cellulose derivative forms usedfor edible films formation: Hydroxy Propyl Cellulose (E463; HPC),Hydroxy Propyl Methyl Cellulose (E464; HPMC), Carboxy Methyl Cellulose(E466; CMC), and Methyl Cellulose (E461; MC). The inherent hydrophilicnature of cellulose derivatives results in poor water vapor barriers andpoor mechanical properties (Gennadios, 1997). Methods of enhancing themoisture barrier of these films would be by the incorporation ofhydrophobic compounds, such as fatty acids into the cellulose ethermatrix to develop a composite film (Morillon V., Debeaufort F., BlondG., Capelle M., Voilley A., 2002. Factors affecting the moisturepermeability of lipid-based edible films: a review. Crit Rev Food SciNutr. Vol. 42, Issue. 1, pp. 67-89).

Chitin/Chitosan

Chitosan is an edible and biodegradable polymer derived from chitin bythe process of deacetylation in the presence of an alkali. It isdescribed in terms of the degree of deacetylation and average molecularweight. Chitosan has poor solubility in neutral solutions but soluble inacids such as acetic, citric and formic acids due to its cationiccharacteristic. It has lots of desirable properties including goodoxygen and carbon dioxide permeabilities, film forming withoutadditives, excellent mechanical properties and antimicrobial activityagainst bacteria, yeasts, and molds. The antimicrobial property ofchitosan is based on the existence of a positive charge on the aminogroup and its attraction to other negatively charged polymers such asthe membrane of microorganisms, cholesterol, and proteins (Muzzarelli,1986). Besides its outstanding antimicrobial properties, chitosan alsoforms semi-permeable coatings, which can modify the internal atmosphere,thus delaying ripening and decreasing transpiration rates in fruits andvegetables (Sandford C., Godwin M., Hardwick P., 1989). Administrativeand Compliance Costs of Taxation. Bath: Fiscal Publications.

Main Types of Lipid-based edible film/coating

Lipid compounds used as edible film consist of acetylatedmono-glycerides, resins and natural wax. Among these compounds, paraffinwax and beeswax are most effective. The hydrophobic characteristic oflipid compounds provides excellent moisture barrier properties, however,due to their poor mechanical properties; they are usually combined withother film forming agents like proteins or cellulose derivatives(Debeaufort, F., Voilley, A., Meares, P., 1994. Water vapor permeabilityand diffusivity through methylcellulose edible films. J. Membr. Sci. No.91, pp. 125-133).

Waxes and Paraffin

Waxes have been used to retard desiccation of citrus fruits in Chinasince the twelfth and thirteenth centuries. The Chinese noted that thewaxes slowed water loss and caused fermentation (Hardenburg R. E. 1967.“Wax and Related Coatings for Horticultural Products. A Bibliography.”Agr. Res. Bull. 15-51, Washington, D.C.: U.S. Dept. of Agric.). Paraffinwax is derived from the distillate fraction of crude petroleum andconsists of a mixture of solid hydrocarbon resulting from ethylenecatalytic polymerization. It contains predominantly straight-chainhydrocarbons with an average chain length of 20 to 30 carbon atoms. Dueto its characteristics such as non-reactive, non-toxic, good moisturebarrier and colorless, it is permitted for use on raw fruits, vegetablesand cheese since the 1930s (Kaplan H. J. 1986. Washing, waxing, andcolor adding. In: Wardowdki W F, Nagy S, Grierson W, editors. Freshcitrus fruit. Westport, Conn.: AVI Publishing Co. 379 p). These coatingsare the most efficient edible compounds blocking transport of moisture,reducing surface abrasion during handling of fruits and controlling softscald formation in apples (Kester J. J., Fennema O. R., 1986. Ediblefilms and coatings: a review. Food Technol Vol. 40, Issue. 12, pp.47-59).

Acetylated Glycerol Monostearate

Acetylation of glycerol monostearate by its reaction with aceticanhydride yields1-stearodiacetin. It is an emulsifier in which aceticacid is bound with monoglyceride. Acetylated monoglycerides (AMG) filmsdisplay the exclusive characteristic of solidifying from the meltingstate to a flexible, wax-like solid. Elongation of the films can be ashigh as 800%, while most lipids in the solid state can be stretched toonly 102%. The films are mainly used for poultry and meat cuts to retardmoisture loss during storage (Bourtoom T., 2008. Edible films andcoatings: characteristics and properties. International Food ResearchJournal, Vol.15, No.3, pp. 237-248). Another application of AMG films isas an antioxidant carrier. When compared to whey protein isolatecoating, an AMG coating makes some of the most effective naturalantioxidants such as tocopherols migrate more freely to the surface dueto the hydrophobic characteristic (Juan I. M., John M. K., 1997. WheyProtein and Acetylated Monoglyceride Edible Coatings: Effect on theRancidity Process of Walnuts. J. Agric. Food Chem. Vol. 45, pp.2509-2513). However, AMG films are shown to have high oxygenpermeability (Hoover M. W., Nathan, P. J., 1981. Influence of tertiarybutylhydroquinone and certain other surface coatings on the formation ofcarbonyl compounds in granulated roasted peanuts. J. Food Sci. Vol. 47,pp. 246-248), therefore they did not provide protection against lipidoxidation in granulated roasted peanuts.

Shellac Resins

Shellac resins are secreted by the insect Laccifer lacca, and arecomposed of a complex mixture of aliphatic alicyclic hydroxyl acidpolymers. It is not recognized as a “GRAS” substance by FDA and as suchis only permitted as an indirect food additive and is mainly used incoatings for the pharmaceutical industry (Berg S., Bretz M., HubbermannE. M., Schwarz K., 2012, Influence of different pectins on powdercharacteristics of microencapsulated antho-cyanins and their impact ondrug retention of shellac coated granulate. Journal of Food Engineering,Vol. 108, No. 1, pp. 158-165). Shellac resins are widely used forcoating citrus and other fruits to enhance their surface glossiness thusdecrease the prevalence of postharvest wilting. However, citrus withshellac resin coating typically has lower internal oxygen, higherinternalcarbon dioxide, and higher ethanol content (an indication of offflavor) than citrus with wax coatings due to their differences in gaspermeance and ability to block openings in the skin. (Bourtoom, 2008).

Composite Edible Film/Coating

Edible films can be synthesized by blending polysaccharides, protein andlipids, which enables one to utilize the advantages of each class offilm (Kester & Fennema, 1984). The combination could be proteins andcarbohydrates, proteins and lipids, carbohydrates and lipids orsynthetic or natural polymers. The aim is to improve the permeability ormechanical properties for specific purposes. The individual componentsof these composite films are blended in the form of an emulsion,suspension, or dispersion of the non-miscible constituents, or insuccessive layers, or in a solution in a common solvent. An example of acomposite polymer widely used in food packaging is polyvinyl acetate. Itis a nontoxic commercially available polymer prepared through emulsionpolymerization and is incorporated with fungicides for protection ofdiverse foods or as a coating for pharmaceutical products(Carmona-Ribeiro A. M., Carrasco L. D., 2013, Cationic antimicrobialpolymers and their assemblies. International Journal of MolecularSciences, Vol. 14, No. 5, pp. 9906-9946). For more than 50 years,techniques such as spraying and dip coating and encapsulation have beenused in the pharmaceutical industries to incorporate bioactive agentswith polymers. For example, an anionic copolymer based on methacrylicacid and methyl methacrylates was used for coating tablets and pills.This coating was resistant to gastric juices but improved the protectionof the tablets against moisture, light and oxygen under tropicalconditions (Petereit H. U., Meier C., Roth E., U.S. Pat. No.: 7,160,558B2, January 2007).

In another example, composite polymer spheres with a sugar coating onthe outside and edible polymer coating inside give them dualfunctionality to target and deliver drugs. The sugar coating providesbarrier to oxygen and gives taste to the tablet, while the ediblepolymer serves as a mechanism for delayed release of the drug. Thepolymer vesicles could be used to mimic a living cell or used as drugdelivery vessels, and could also be used to convey drugs andbiomolecules to injured or cancerous tissues in animals or humans(Schlaad H., You L., Sigel R., 2009, Glycopolymer vesicles with anasymmetric membrane. Chemical Communications, No. 12, pp. 1478-1480).

The invention has been shown and described herein in what is consideredto be the most practical and preferred embodiment. The applicantrecognizes, however, that departures may be made therefrom within thescope of the invention and that obvious modifications will occur to aperson skilled in the art. The examples which follow are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention. All references cited herein are hereby incorporated intheir entirety by reference.

EXAMPLE 1

This example is directed to developing the potential lead narrowspectrum small molecule inhibitors of Campylobacter jejuni for oraladministration of chickens to control Campylobacter with ultimate goalof reducing human infections. This will be accomplished via theevaluation and optimization of the derivatives (TH-4 and TH-8)identified though a high throughput screen (HTS) of over 4,182pre-enriched compounds (see below). Our long term goal is to improvefood safety by reducing the transmission of Campylobacter to humans viathe food chain, through development of specific, orally compatible,water soluble narrow spectrum small molecule inhibitors. The three aimsof this experiment are:

-   -   Aim 1. Explore and improve the antibacterial activity and        aqueous solubility of the lead compounds TH-4 and TH-8 through        structural modification focused on SAR and lead optimization        studies.    -   Aim 2. Assess the efficacy of TH-8 and TH-8 derivatives in vitro        and determine the effect on broiler gut health and C jej uni        colonization.    -   Aim 3. Identify target and explore mechanisms of action of most        potent small molecule inhibitors.

For a discussion of the high throughput process see Kumar et al.(2016)“Novel anti-campylobacter compounds idenfied using high throughputscreening of pre-selected enriched small molecules library” Frontiers inMicrobiology, Vol. 7, Article 405 pp. 1-12. In our recent study, wescreened a small molecule library of 4,182 compounds against highlypathogenic C. jejuni 81-176 strain (Wallace, I. M., M. L. Urbanus, G. M.Luciani, A. R. Burns, M. K. Han, H. Wang, K. Arora, L. E. Heisler, M.Proctor, R. P. St Onge, T. Roemer, P. J. Roy, C. L. Cummins, G. D.Bader, C. Nislow, and G. Giaever. 2011. Compound prioritization methodsincrease rates of chemical probe discovery in model organisms. Chemistry& biology 18:1273-1283).

TABLE 1 Assessing the anti-camylobacter activity for two re-synthesizedcompounds using synthetic films and pathogenic C. jejuni 81-176 strains.% of compound Diameter of zone of Films incorporation inhibition Filmwith no compound — No inhibition Film with JA-144 1.0 1.2 cm Film withJA-128 1.0 2.7 cm

Seven hundred and eighty one compounds were identified possessingbactericidal or bacteriostatic property against C. jejuni at 100 μMconcentrations (Kumar et al 2016) (FIG. 2) (Kumar A, Drozd M,Pina-Mimbela R, Xu X, Helmy Y A, Antwi J, Fuchs J R, Nislow C, TempletonJ, Blackall P J, Raj ashekara G. (2016) Novel Anti-CampylobacterCompounds Identified Using High Throughput Screening of a Pre-selectedEnriched Small Molecules Library. Front Microbiol. Apr 6;7:405. doi:10.3389/fmicb.2016.00405. eCollection. PMID: 27092106). Further throughsecondary screens we identified several compounds with narrow spectrumactivity specific to C. jejuni and C. coli. We synthesized 15derivatives of one selected hit (Compound 1) (FIG. 3) from primarystreening and tested for specificity and potency against diversefoodborne pathogens like Salmonella, E. coli, Listeria and also severalcommensal and/or probiotic bacteria. Two derivatized compounds (TH-4(1d) and TH-8 (le) found to be specific to C. jejuni and C. coli andpossessed low MICs (1.2 5 μM). Both TH-4 and TH-8 lacked toxicityagainst the colon cells (CCD-112CoN, ATCC) (data not shown) and alsodisplayed promising in vivo results in a chicken model (FIG. 4) whenadministered orally. These two compounds also retained the in effectagainst both Campylobacter species when incorporated in edible filmsuggesting that this compound can also be exploited for post-harvestCampylobacter control also (eg. in the form of meat wrapper), However,these compounds were not readiliy soluble in water and were administeredto chicken in 30% DMSO. To be feasible for mass application in poultryproduction the compounds need to be water soluble. These two compoundswill be used and subjected to further structural studies to develop themas specific narrow spectrum water soluble compounds for application forchickens. Though, beyond scope of this proposed study, we plan todevelop these compounds for future application in humans also. This isexpected to lead to timely development of innovative strategy to reduceCampylobacter in poultry, a natural host for C. jejuni and willsignificantly impact food safety there by reducing humancampylobacteriosis cases.

Aim 1. Explore and Improve the Antibacterial Activity and AqueousSolubility of the Lead Compounds TH-4 And TH-8 Through StructuralModification Focused on SAR and Lead Optimization Studies.

Compound 1 was identified via a screening of 4,182 compounds for growthinhibition against C. jejuni. This compound was re-synthesized accordingto FIG. 5 for validation purposes. As shown in FIG. 2, a total of 15analogues of hit 1 were then prepared as a part of a preliminaryhit-to-lead campaign to establish whether this class of compounds woulddisplay useful drug properties. Compounds TH-4 (1d) and TH-8 (1e) wereshown to display the best combination of potency against the bacteria,lack of toxicity against the colon cells and promising in vivo resultsin a chicken model as described above and thus have been adopted aspromising lead compounds for further structural study and development.The prime advantage of this structural scaffold is the ability toprepare a wide array of structurally diverse compounds in essentially asingle transformation from commercially available precursors.Derivatization will focus on two key aspects: 1) expanding thestructural diversity of the analogues for structure-activityrelationship (SAR) studies and 2) the rational design of more watersoluble analogues for translational studies. The initial SAR studieswill involve the synthesis of a much larger library of compounds thattarget modifications of both the thiophene portion of the molecule andthe benzylamine. Utilizing commercially available sulfonyl chloridederivatives number of, a thiophenes and isosteric aryl and heteroarylrings will be explored, including those containing polar heteroatoms andfunctional groups. The substitution of the benzylamine, both in a stericand electronic sense, will also be addressed. Once the SAR has beenestablished, the focus of the project will shift towards the improvementof water solubility of these agents utilizing data obtained from thislibrary. The ACD/Labs Percepts Suite will be used to predict therelative solubility (LogD) for the compounds as well as to aid in thedesign of more water soluble compounds. All compounds prepared duringthe course of these studies will be provided to Dr. Rajashekara forbiological evaluation and that data will be incorporated into subsequentompound design, creating an iterative cycle of drug discovery anddevelopment.

Aim 2. Assess the Efficacy of TH-8 and TH-8 Derivatives in vitro andDetermine the Effect on Broiler Gut Health and C. jejuni Colonization.

Selected derivatives will be screened in number invitro assay asdescribed previously and in our recent paper .(n fs); 1) MinimumInhibitory Concentration (MIC), 2) minimum bactericidal effect, 3)effect on diverse C. jejuni strains, 4) cytotoxicity to human intestinalcells, 5) effect on intracellular survival of C. jejuni in culturedintestinal cells, 6) The impact of small molecules on acquisition ofresistance by Campylobacter.

In vivo evaluation of small molecules: We will test the applicability ofthe selected compounds to specifically reduce the Campylobacter load inbroiler chickens just before slaughter. Throughout the experiment,chickens will receive the same type of commercially available crumblefeed. For this, we will inoculate xx groups of 5 week old broilerchickens (n=10) free of Campylobacter with C. jejuni (lx 10⁶ cfu/chickin 200 μI of PBS). Group 1 will receive the compounds at the determinedMIC level (from above): Group 2 will receive 10 times the MIC; and Group3 will serve as untreated control. Three days following C. jejuniinoculation, these chickens will be given water with or without smallmolecule inhibitors daily for 1 week. Following treatment (at marketage; −42 days) hickens will be killed and kinetics of colonization inthe cecum will be monitored by determining CFU. In addition, we willalso monitor faecal shedding following small molecule administrationdaily until the termination of the experiment. For those compounds thatexhibit significant reduction in C. jejuni numbers, we will repeat theexperiment to confirm our findings.

In addition, other important parameters, such as average feed intake,average gain, feed conversion ratio, and mortality will be recorded.Further, we will conduct metagenomic studies on the cecal microflorafrom chickens treated with small compounds and compare to controluntreated birds (Turnbaugh et al 2009). This will provide a morecomprehensive picture on the effect of these compounds on the gutmicroflora

Our future studies will focus on testing compounds that give the bestresult in our small pen trials, under near to commercial productionsetting.

Aim 3. Identify Target and Explore Mechanisms of Action of Most PotentSmall Molecule Inhibitors.

In previous work, analysis of a EZ::TN transposon-based mutant librarycomprised of 7,201 individual mutants (4.48><coverage of the genome)revealed 195 essential gene candidates (Smith et la 2009 Quantitativephenotyping via deep barcode sequencing. Genome Res. 2009Oct;19(10):1836-42. doi: 10.1101/gr.093955.109. Epub 2009 Jul 21; Smithet al 2012 Barcode sequencing for understanding drug-gene interactions.Methods Mol Biol. 2012;910:55-69. doi: 10.1007/978-1-61779-965-5_4) Wehave recently developed a similar high-density universal BarcodedTransposon-sequencing approach (BarT-seq) (Smith et al 2012) that can bereadily adapted to diverse Campylobacter species and strains. In thisassay, each strain carries a precise start to stop deletion is“barcoded” with two unique 20 base pair sequences that serve as strainidentifiers. These pools are grown competitively in any condition toidentify genes most important for growth. In practice strains carryingdeletions of these genes become depleted from the pool over time. Therelative abundance of each strain is measured by the abundance of thebarcodes. Specifically, following pooled cell growth, genomic DNA isextracted from cells, barcodes are PCR amplified using primers common toevery strain, and relative strain abundance quantified. The advantagesof our BarT-seq approach are several-fold. First, after initialconstruction of the mutant library (with a minimum genome coverage ofSOX) containing 10-20 insertions/gene, we can unambiguously identify thelocation of each transposon insertion in a single deep coveragesequencing run. Once each barcode is assigned to a genomic location,subsequent genome-wide screens require only minimal sequencing to“count” each unique barcode. This allows for the sampling of the sameexact transposon library in hundreds to thousands of conditions. Byvirtue of this throughput, we can assess the genes required for survivalin diverse conditions, including but not limited to, i) wide ranges ofanti-Campylobacter concentrations, ii) assessing a matrix of drugcombinations, and iii) assessing the requirements of each gene in thepresence and absence of drug in vivo in chickens over time.

In parallel to the BarT-seq approach to define the drug-geneinteractions that underlie the efficacy of our novel anti Campylobacteragents and its derivatives, we will employ our well-established yeastHIPHOP chemogenomic assay (Lee A Y, et al. 2014. Mapping the cellularresponse to small molecules using chemogenomic fitness signatures.Science. Apr 11;344(6180):208-11. doi: 10.1126/Science. 1250217. Erratumin: Science. 2014 May 23;344(6186):1255771). HIPHOP provides directmechanistic insight into a gene's requirement for cell growth andviability by combining small-molecule screening with genomic targetidentification. Because the screens are both target and drug agnostic,one obtains the whole-cell response to perturbation In our recent study(Lee et al., 2014), we systematically characterize the cellular responseto small molecule perturbation by screening 3250 compounds using ourHaploinsufficiency Profiling (HIP) and Homozygous Profiling (HOP)chemogenomic platform. HIP exploits drug-induced haploinsufficiency, agrowth or fitness defect (ED observed in a heterozygous strain deletedfor one copy of the drug's target. By measuring the drug-induced FDs of−1,100 heterozygous strains representing the yeast essential genome,strains exhibiting the greatest FDs identify candidate protein targets.In the complementary HOP assay of −4,800 homozygous deletion strains,nonessential genes required to buffer the targeted pathways can beidentified. Each combined HIPHOP profile provides a genome-wide view ofthe cellular response to a specific compound . See FIG. 6

Data analysis and expected results. We anticipate identifying highlypotent water soluble lead compounds having specific narrow spectrumeffect on C. jejuni. Through in vivo studies, we anticipate to identifycompounds having promising effect on C. jejuni when administered orallyin water, a prerequisite in poultry production. The optimization ofthese lead compounds will continue throughout the course of the proposedgrant and promising orally active compounds should be identified priorto the completion of the funding period, ultimately facilitating furthergrant applications for clinical/commercial testing. Data generated usingseveral biological assays (both in vitro and in vivo) in this study willbe analyzed using T-test or one-way ANOVA followed by the Tukey'spost-test P<0.01 or 0.05 (a level) will be considered significant.

Innovation. Despite the appreciated magnitude of the food safety hazardassociated with Campylobacter, effective and practical interventions arescarce for either pre harvest or post harvest control. Our proposedresearch will yield new pre harvest intervention strategy thus will havedirect impact on food safety, public health, and global food policy. Theplanned activities utilize multidisciplinary approaches and will beconducted by a highly qualified team of investigators who have strongexpertise in Camplobacter and food safety , medicinal chemistry andchemogenomics. Our study is innovative and takes a novel approach ofidentifying narrow spectrum small molecule inhibitors of Campylobacter.Although the amount of genomic information on both pathogens and theirhosts is growing, drug remedies remain as elusive as ever. Thischallenge is greatest for antimicrobials, in large part becausepharmaceutical companies have abandoned their efforts in this area dueto its very challenging pharmaco-economics. To directly address thechallenge of increasing food safety, here we will develop specific,orally bioavailable narrow spectrum small molecule inhibitors ofCampylobacter. Because of the small size, solubility and stability underadverse environmental conditions, they are highly suitable for massapplication, pre-requisite in the poultry production.

Relation to Enteric CETR. Reducing Campylobacter infections in humanswill significantly reduce the burden on the health system. Sincemajority of human infections are due to consumption of contaminatedpoultry meat and meat products, on farm control of Campylobacter willhave significant impact on the bacterial load in the processed foodwhich should translate to reduced human infections. It is estimated thatpre-harvest reduction of Campylobacter by 2 logs or higher could resultin up 90% reduction in human campylobacteriosis. In addition, foodsafety is a major challenge for the poultry industry both nationally andinternationally. Ensuring safe food supply is critical for havingpositive influence on the consumers thereby enabling the growth ofpoultry industry.

Results of the primary screening can be found in FIGS. 6-15. In FIG. 6 agraph is shown. As described in earlier primary screening, we screened16 compounds for C. jejuni growth inhibition. A cut off percentage valueof 70.0% growth inhibition was used to identify potent compounds. SeeFIG. 7. We also screened 15 derivatized compounds for C. coli growthinhibition. A cut off percentage value of 70.0% growth inhibition wasused to identify potent compounds. Based on both C. jejuni and C. coligrowth inhibition assay, we selected four derivatized (TH4, TH8, THI 1and TH13) compounds (highlighted in red box) for further analysis. SeeFIGS. 8A and B. Testing the effect of TH-4 at two differentconcentrations on the growth of C. jejuni and C .coli. Where positivecontrol (P CNTL) include Campylobacter culture with know antibiotics andnegative control (N CNTL) include Camylobacer culture alone. See FIGS.9A and B. Testing the effect of TH-8 at two different concentrations onthe growth of C. jejuni and C. coli. Where positive control (P CNTL)include Campylobacter culture with known antibiotics and negativecontrol (N CNTL) include Camylobacter culture alone. See FIG. 10.Testing the efficacy of TH4 and TH8 compounds when incorporated inedible films against C. jejuni and C. coli. Where control include ediblefilm without any incorporation. Both TH4 and TH8 were effective inreducing Campylobacter by 4-6 log. See FIG. 11.

As described in primary HTS, we screened re-synthesized two compoundsfor C. jejuni 81-176 growth inhibition. Both re-synthesized compoundseffective inhibiting Campylobacter growth at tested concentrations.FIGS. 12A and B shows texting the effect of re-synthesized compoundJA-144 at two different concentrations on the growth of C. jejuni and C.coli. Where positive control (P CNTL includes Campylobacter culture withknown antibiotics and negative control (N CNTL) include Camylobacterculture alone. See FIG. 13. Testing the efficacy of JA-144 whenincorporated in edible films against pathogenic E.coli, Listeria strain,C. jejuni and e. coli bacteria. The control (CNTL) include edible filmalone. JA-144 was effective in reducing most of the pathogenic bacteriaby 4 log. See FIG. 14. A cytotoxicity assay for potent semi-syntheticderivative compounds were performed using normal colon cells andsulfohodamine B methods. See FIG. 15. A cytotoxicity assay was performedfor two re-synthesized compounds using normal colon cells andsulforhodamine B methods.

EXAMPLE 2 Abstract

This study investigated the antimicrobial activities of small moleculecompounds (JA-144, TH-4, and TH-8) against E. coli K12, Listeriainnocua, Campylobacter jejuni and Campylobacter coli. Minimum InhibitoryConcentrations (MIC) for JA-144 were 100 ppm against E. coli K12, 50 to100 ppm against L. innocua at 37° C., and 3.125 to 6.25 ppm for C.jejuni and C. coli at 42° C., respectively. TH-4 and TH-8 wereineffective against E. coli K12 and L. innocua. The MICs for TH-4 were12.5 to 25 ppm against C. jejuni and C. coli at 42° C., while for TH-8they were 25 to 50 ppm. Growth curves for E. coli K12, L. innocua, andC. jejuni exposed to JA-144, TH-4 and TH-8 at their MICs showed thatJA-144 completely inhibited E. coli K12 and L. innocua at 100 ppm and C.jejuni at 6.25 ppm, respectively, TH-4 completely inhibited C. jejuni at25 ppm while TH-8 accomplished it at 50 ppm. When incorporated intodried tapioca films, efficacy losses for JA-144 were 43.48%, 36.42%,0.25% and 2.03%, respectively for E. coli K12, L. innocua, C. jejuni andC. coli. TH-4 losses were 6.09% and 29.02%, respectively, while for TH-8they were 25.52% and 48.01%, respectively, for C. jejuni and C. coli.

We have screened a pre-selected bioactive small molecule library of4,182 compounds against highly pathogenic C. jejuni 81-176 strain. Theobjectives of this study were to investigate the antimicrobialactivities of synthesized small molecules (JA-144, TH-4 and TH-8) (FIG.1), identified from our earlier screen, dissolved in Dimethylsulfoxide(DMSO), and incorporated into tapioca edible films. Antibacterialproperties were tested against E. coli K12 and Listeria innocua,Campylobacter jejuni and Campylobacter coli. The E. coli K12 and L.innocua were used as bacterial surrogates of E.coli O157:H7 and L.monocytogenes, respectively.

This structural scaffold of these compounds are amenable to preparing anarray of structurally diverse compounds in essentially a singletransformation from commercial precursors.

2. Materials and Methods 2.1. Materials

Tapioca starch powder was purchased from a local supermarket inColumbus, Ohio. It was irradiated at the Ohio State University NuclearReactor Laboratory in order to achieve sterilization. This was used asthe main ingredient in the films. Distilled water was used to make asuspension of the starch powder. Glycerol≥99.0% (Sigma-Aldrich®) wasobtained from Fisher Scientific (Fisher Scientific, Fair Lawn, N.J.) andused as a plasticizer. Acetic Acid (ACS Reagent≥99.7%), DimethylSulfoxide (ACS Reagent≥99.9%) and 2,3,5-Triphenyltetrazolium Chloride(≥95.0%) were purchased from Sigma-Aldrich® and used as a solvent andbacterial dye agent, respectively. E. coli K12 and Listeria innocua werepurchased from the American Type Culture Collection (Manassas, Va.).Campylobacter jejuni and Campylobacter coli were obtained from Dr.Rajashekara's laboratory, Wooster, Ohio. Tryptic Soy Broth (TSB) andTryptic Soy Agar were purchased from Difco (Sparks, Md.). Mueller HintonAgar, Mueller Hinton Broth and Fisher BioReagents Peptone (Granulated)were purchased from Thermo Scientific®, while 1.5 ml Micro CentrifugeTubes, 96-Well Microplates, 100 mm Petri Dishes were obtained fromThermo Scientific®, respectively. The synthesized small moleculesreferred to as JA-144, TH-4 and TH-8 were obtained from Dr. James Fuch'slaboratory at the College of Pharmacy, OSU. The chemical structures ofthese molecules are shown in FIG. 1.

2.2. Method 2.2.1. MIC Tests for Small Molecule Compounds. 2.2.1.1.Preparation of Bacterial Culture.

E. coli K12, L. innocua, C. jejuni and C. coli were used in the assay.The bacteria were maintained on nutrient agar slants at 4° C. Beforeuse, a single colony of each bacterium was transferred into 50 ml tubescontaining MHB using aseptic techniques. The tubes were capped andplaced in an incubator overnight at 37° C., aerobically (for E. coli andL. innocua), and at 42° C. (for C. jejuni and C. coli),microaerobically. After 24 hours incubation, the broths were centrifugedat 4000 rpm for 5 min using appropriate aseptic precautions. Thesupernatants were discarded and the pellets resuspended using 20 ml ofsterile peptone water and centrifuged again at 4,000 rpm for 5 min. Thisstep was repeated until the supernatants were clear. The pellets werethen suspended in 20 ml of sterile peptone water and labeled as StandardBroth (SB). As for E. coli K12, and L. innocua, six consecutive 10-folddilutions were made to reach the initial inoculum of 10³ CFU/ml. For C.jejuni and C. coli, the initial inoculum was obtained by measuring theoptical density of the SB at 595 nm, and serial dilutions carried outuntil the optical density reached 0.05, which was equal to 10⁶ CFU/ml.

2.2.1.2 Preparation of Small Molecule and Other Stock Solutions.

The small molecule solutions were prepared by dissolving 100 mg of eachcompound at room temperature in 1 ml DMSO in separate test tubes toreach a concentration of 100,000 ppm. A vortex mixer was used to ensurethat each compound was well dissolved into a homogenous solution. Thesmall molecule stock solutions were then stored at −20° C.

The 2,3,5-Triphenyltetrazolium Chloride (TTC) stock solution wasprepared by dissolving 20 mg in 1 ml distilled water at room temperatureto reach a concentration of 20,000 ppm. A vortex mixer was used toensure that it was well dissolved. The TTC stock solution was stored at−20° C. for future use.

The range of concentrations for the small molecules in the filmsdepended on previous cytotoxicity assay conducted by Dr. EsperanzaCarcache de Blanco's laboratory in the College of Pharmacy at OSU. Thistest determined the highest concentrations of each compound to causelysis of human colon cells. The results showed that the values forJA-144, TH-4 and TH-8 were 200 μM. When converted to ppm, they were 66.9ppm, 61.1 ppm, 60.4 ppm, respectively. The survival rates of colon cellsfor JA-144, TH-4 and TH-8 at 200 μM were 99%, 60%, and 79%,respectively. Since JA-144 showed less toxicity on human colon cells,the starting concentration for this study was 100 ppm; while for TH-4and TH-8, the starting concentration were 50 ppm, respectively.

2.2.1.3. Preparation of the Plates for MIC Testing.

A sterile 96 well plate was used for all MIC tests. A positive controlwas used in this study. This positive control was made of 198.6 μl ofMHB with E. coli K12, L. innocua, C. jejuni, and C. coli at aconcentration of 10⁶ CFU/ml, 0.4 μl of Chloramphenicol (2 μl/ml inbroth), and 1 μl TTC stock solution (100 ppm in broth). This waspipetted into the first column of the plate. The MIC test groups werethen placed in row 2 to row 11, plus 197 μl of MHB inoculated with E.coli K12, L. innocua, C. jejuni, or C. coli at 10⁶ CFU/ml, 2 μl of DMSOwith diluted JA-144, TH-4 or TH-8, and 1 μl TTC stock solution. Anegative control group was prepared by repeating the above but withoutJA-144, TH-4 or TH-8. This negative control group was pipetted into thelast column of the plate. Triplicate tests were done for each bacterialstrain. The 96-well micro plates with E. coli K12 and L. innocua wereincubated at 37° C. aerobically for 24 hours; while the plates with C.jejuni and C. coli were incubated at 42° C. microaerobically for 24hours, respectively. The MIC was the lowest concentration of the agentthat completely inhibited visible growth of the test microorganisms. TheMIC values depended on a color change. No color change was observed whengrowth of the bacteria was inhibited.

TABLE 1 Composition of Tapioca Film on Wet and Dry Percent Weight Basis.Wet basis Dry basis Composition Weight % Composition Weight % Tapiocastarch  5% Tapioca starch 72.78% Glycerol 1.8% Glycerol 26.20% Aceticacid 0.7% Small molecules  1.02% Small molecules 0.07%  Distilled water93.23%  Total 100%  Total  100%

2.2.1.4. Growth Curves Bacteria Against all Test Compounds at the MICValues.

For E. coli K12 and L. innocua, the bacterial incubations were followedby measurements of the Optical Density (OD) values every 15 minutes inan automated Sunrise™ Tecan Spectrophotometer (Tecan Co.) during anincubation period of 24 hours at 37° C. For C. jejuni, the incubationexperimental design was similar, but the measurement period occurredevery 4 hours. From the data collected, comparisons were made with thecontrol samples and statistical analyses were used to determine thesignificance between the means for all observations.

2.2.2. Preparation of Tapioca Films.

The film forming solutions were prepared using blends of tapioca starchand JA-144, TH-4 and TH-8 dissolved in acetic acid with glycerol as aplasticizer. This began when aliquots of 100 mg of each small moleculecompound was first dissolved in 1 ml acetic acid. Tapioca starch (5%w/w) and acetic acid solutions (0.7% w/w) with the small molecules and1.8% w/w glycerol were dissolved into 100 ml distilled water as shown inTable 2.

TABLE 2 A summary of Log Reduction Tests for E. coli K12 and L. innocua.E. coli K12 L. innocua In JA-144 2.96 ± 0.30^(a) 3.14 ± 0.11^(b) BrothCNTL 8.81 ± 0.15^(a) 8.66 ± 0.09^(b) Log₁₀ CFU/ml reduction 5.86 5.52 InJA-144 5.58 ± 0.08^(a) 5.22 ± 0.07^(b) Film CNTL 8.90 ± 0.13^(a) 8.73 ±0.29^(b) Log₁₀ CFU/ml reduction 3.31 3.51 Percent efficacy loss due toincorporation  43.48%  36.42% Values within the same row with the sameletters are not significantly different (p > 0.05).

All dispersions were heated in a water bath (70° C.) for 15 min withstirring until completely gelatinized. An Ultrasonic Sonicator(Graymills Co., Chicago, Ill.) was used to remove air bubbles from thegelatinized solutions. The edible films were prepared by casting thesolutions (107 g) into 10-inch radius Teflon plates. These were ovendried at 45±2° C. for 12 hours, then the dried films peeled off from theplate surfaces. The final concentration of each small molecule compoundin the dry film was approximately 1% w/w. The film thicknesses weremeasured using a Magna-Mike 8500 Thickness Gage (Olympus, Japan), withresolution of 0.001 mm. A total of 5 measurements per film, at variouslocations were taken to determine the average thickness.

2.2.3. Inoculation and Testing of Dry Tapioca Films against TestMicroorganisms.

The films prepared above were cut into small pieces that were capable offitting into 1.5 ml micro-tubes. The weight of each film sample rangedfrom 5 to 15 mg. The micro-tubes were then exposed to UV light for 18hours to eliminate possible contamination that may have occurred duringthe cutting and transferring process. The amount of bacterial culture ineach tube depended on two factors: the amount of small molecules in thefilm, and the MIC value that completely inhibited the bacterial growth.After adding the proper amount of bacterial culture, the micro-tubeswere placed in an incubator aerobically at 37° C. (for E. coli and L.innocua), or microaerobically at 42° C. (for C. jejuni and C. coli), for24 hours, respectively. The antimicrobial activity tests using tapiocastarch films incorporated with the JA-144, TH-4 or TH-8 were followed byMIC and bacterial growth curve tests. Preparation of the bacterialcultures was the same as for the MIC tests.

To determine the test microbial growth kinetics, Mueller Hinton Agar(MHA) was used as a growth medium. A 4% w/w solution was prepared bydissolving 40 g MHA powder in 1,000 ml distilled water and thensterilized at 121° C. for 25 min. It was then transferred to a 50° C.water-bath and allowed to cool. Afterwards, it was poured intopre-labeled sterile Petri dishes on a level surface at room temperature(23° C.) and dried so that no drops of moisture remained on surface ofthe agar.

2.2.4. Inoculation of Plates

After 24 hours incubation, the broth was centrifuged at 4,000 rpm for 5min at 23° C. with appropriate aseptic precautions. The supernatant wasdiscarded and the pellet resuspended using 1 ml of sterile peptone waterand centrifuged again at 4000 rpm for 5 min. This step was repeateduntil the supernatant was clear. The pellets were then suspended in 1 mlsterile peptone water, and seven consecutive 10-fold dilutions were madeto obtain inoculums ready for the plate counts. For the experimentalgroups with the small molecule compounds, 0.1 ml of diluted broth(dilution of 10⁻²; 10⁻⁴; 10⁻⁶) were inoculated on to the plates. For thecontrol groups, 0.1 ml of diluted broth (dilution of 10⁻⁶ and 10⁻⁷) wasadded to the plates. They were then incubated at 37° C. aerobically (forE. coli and L. innocua), or 42° C. microaerobically (for C. jejuni andC. coli) for 24 hours, and then colony numbers on each plate countedusing a Darkfield Colony Counter manufactured by American Optical(Buffalo, N.Y.).

2.3. Statistical Analysis.

All experiments were performed in triplicate and differences in themeans analyzed by a SAS statistical program. One-way analysis ofvariance (ANOVA) was carried out to evaluate significance in differences(p<0.05) between the influences of the small molecules on the microbialgrowth.

3. Results and Discussion 3.1 MIC Tests of JA-144, TH-4 and TH-8.

The MIC of JA-144 against E. coli K12 was 100 ppm, while the result forL. innocua, ranged from 50 ppm to 100 ppm. The MIC of JA-144 against C.jejuni and C. coli ranged from 3.125 ppm to 6.25 ppm, respectively. Theresults showed that TH-4 had no antimicrobialactivity against E. coliK12 and L. innocua. However, the MIC of TH-4 against C. jejuni and C.coli ranged from 12.5 ppm to 25 ppm, respectively. TH-8 also did notshow antimicrobialactivity against E. coli K12 and L. innocua, but forC. jejuni and C. coli, the MIC ranged from 25 ppm to 50 ppm,respectively.

3.2 Bacterial Growth Curves of Test Microorganisms against SmallMolecules at MIC.

FIG. 17 shows the growth curve results for E. coli K12 exposed tovarying concentrations of JA-144. At 100 ppm concentration level, thegrowth of E. coli K12 was not detected. The 50 ppm was least effectivebut still inhibited the growth of E. coli K12 more than that of thenegative control. Similar results were obtained for JA-144 against L.innocua (FIG. 18). For C. jejuni treated with JA-144, FIG. 19 shows thatthe growth of the organism was inhibited more by the 6.25 ppm treatmentwhen compared with the 3.125 ppm. The results also show that the 6.25ppm treatment had a greater impact when compared with the positivecontrol. A similar result was also obtained for C. jejuni treated withTH-4 at 25 and 12.5 ppm (FIG. 20). In that case, the 25 ppm treatmentwas more effective than the positive control. For the TH-8 tests, asshown in FIG. 21, the results show that the growth of C. jejuni wascompletely inhibited by a concentration of 50 ppm.

3.3 Antimicrobial Tests of JA-144 in Growth Medium against E. coli K12and L. innocua.

FIGS. 22A and B shows the effect of 100 ppm JA-144 (in the broth) on E.coli K12 and L. innocua. The results show significant reductions(p<0.05) of 5.86 and 5.52 log₁₀ CFU/ml, respectively after 24 hoursincubation when compared with the control. For C. jejuni and C. coli inthe broth, JA-144 at 6.25 ppm and 3.125 ppm concentrations resulted insignificant reductions (p<0.05) of 6.35 and 6.27 log₁₀ CFU/ml,respectively after 24 hours incubation (FIGS. 23A and B), when comparedwith the control.

For TH-4 at 25 ppm and 12.5 ppm concentrations against C. jejuni and C.coli in broth, the results show significant reductions (p<0.05) of 6.37and 6.16 log₁₀ CFU/ml, respectively after 24 hours incubation (FIGS. 23Aand B), when compared with the control.

When C. jejuni and C. coli in the broth were treated by 50 ppm TH-8, theresults showed significant differences (p<0.05) of 5.39 and 5.51 log₁₀CFU/ml, respectively after 24 hours incubation (FIGS. 23A and B), whencompared with the control. All log reduction-testing results aresummarized in Tables 3, 4 and 5.

TABLE 3 A Summary of Log Reduction Tests for C. jejuni and C. coli. InBroth C. jejuni Log₁₀ CFU/ml C. jejuni Control reduction JA-144 2.90 ±0.09^(a) 9.25 ± 0.01^(ab) 6.35 TH-4 2.78 ± 0.09^(a) 9.15 ± 0.06^(ab)6.37 TH-8 3.64 ± 0.05^(a) 9.03 ± 0.08^(ab) 5.39 C. coli Log₁₀ CFU/ml C.coli Control reduction JA-144 2.93 ± 0.26^(a) 9.20 ± 0.02^(ab) 6.27 TH-43.21 ± 0.12^(a) 9.38 ± 0.04^(ab) 6.16 TH-8 4.11 ± 0.05^(a) 9.62 ±0.04^(ab) 5.51 Values within the same row with the same letters are notsignificantly different (p > 0.05).

TABLE 4 A Summary of Log Reduction Tests for C. jejuni and C. coli.Values within the same row with the same letters are not significantlydifferent (p > 0.05) In Broth C. jejuni Log₁₀ CFU/ml C. jejuni Controlreduction JA-144 2.90 ± 0.09^(a) 9.25 ± 0.01^(ab) 6.35 TH-4 2.78 ±0.09^(a) 9.15 ± 0.06^(ab) 6.37 TH-8 3.64 ± 0.05^(a) 9.03 ± 0.08^(ab)5.39 C. coli Log₁₀ CFU/ml C. coli Control reduction JA-144 2.93 ±0.26^(a) 9.20 ± 0.02^(ab) 6.27 TH-4 3.21 ± 0.12^(a) 9.38 ± 0.04^(ab)6.16 TH-8 4.11 ± 0.05^(a) 9.62 ± 0.04^(ab) 5.51 Values within the samerow with the same letters are not significantly different (p > 0.05).

TABLE 5 A Summary of Log Reduction Tests for C. jejuni and C. coli. InFilm C. jejuni % Efficacy Log₁₀ CFU/ml loss due to C. jejuni Control.reduction incorporation JA-144 2.86 ± 0.13^(a) 9.22 ± 0.05^(ab) 6.360.25 TH-4 3.16 ± 0.14^(a) 9.15 ± 0.07^(ab) 5.99 6.09 TH-8 5.10 ±0.07^(a) 9.11 ± 0.12^(ab) 4.01 25.52 C. coli % Efficacy Log₁₀ CFU/mlloss due to C. coli Control. reduction incorporation JA-144 2.93 ±0.16^(a) 9.07 ± 0.05^(ab) 6.14 2.03 TH-4 5.03 ± 0.07^(a) 9.40 ±0.04^(ab) 4.37 29.02 TH-8 6.83 ± 0.06^(a) 9.70 ± 0.07^(ab) 2.87 48.01Values within the same row with the same letters are not significantlydifferent (p > 0.05)3.4 Antimicrobial Activity Tests of all Compounds Incorporated intoTapioca Films.

For E. coli K12 and L. innocua, 1% w/w of JA-144 film resulted insignificant reductions (p<0.05) of 3.31 and 3.51 log₁₀ CFU/ml,respectively after 24 hours incubation (FIG. 22B), when compared withcontrol. The percentages of antimicrobial activity lost due toincorporation of JA-144 into the tapioca films were 43.48% and 36.42%,respectively. For C. jejuni and C. coli, JA-144 at 6.25 ppm and 3.125ppm resulted in significant reductions (p<0.05) of 6.36 and 6.14 log₁₀CFU/ml, respectively after 24 hours incubation (FIGS. 24A and B), whencompared with control. Percentages of antimicrobial activities lost dueto incorporation of JA-144 into the tapioca films were 0.25% and 2.03%,respectively.

For TH-4 at 25 ppm and 12.5 ppm against C. jejuni and C. coli, theresults show significant reduction (p<0.05) of 5.99 and 4.37 log₁₀CFU/ml, respectively after 24 hours incubation (FIG. 24A), when comparedwith control. Percentages of antimicrobial activities lost when TH-4 wasincorporated into the films were 6.09% and 29.02% for C. jejuni and C.coli, respectively.

When C. jejuni and C. coli were treated by TH-8 at 50 ppm, thereductions in growth were significant (p<0.05) at 4.01 and 2.87 log₁₀CFU/ml, respectively after 24 hours incubation (FIGS. 24A, B), whencompared with control. The reductions in antimicrobial activities causedby incorporating TH-8 into the films were 25.52% and 48.01% for C.jejuni and C. coli, respectively. All log reduction-testing results aresummarized in Table 4 and Table 5.

4. Discussion 4.1 Minimum Inhibitory Concentration (MIC) Tests forJA-144, TH-4 and TH-8.

It is to be noted that MIC values for JA-144 against C. jejuni and C.coli were significantly lower than for E. coli K12 and L. innocua,suggesting that Campylobacter spp. was more sensitive to JA-144. Thiscould be explained by the fact that aerobic bacteria grow 30 to 50 timesfaster than microaerobic bacteria (Mosen, 2006). This faster growth rateof aerobic bacteria is indicative of their greater resistance to drugs,when compared with microaerobes (Diana et al., 1991). The results alsorevealed that JA-144 inhibited all tested bacteria, while TH-4 and TH-8were only effective against the Campylobacter species. This could beattributed to the polar hydroxyl group in the JA-144 molecule. Rotundaet al., (2010) reported that hydroxyl groups could disrupt the integrityof biological membranes by binding to the phospholipid bilayerhydrophobic core, solubilizing membrane-associated proteins, and finallycausing membrane breakdown and lysis of the bacterial cell membrane.

4.2 Growth Curve Measurements Using JA-144, TH-4, and TH-8 as Inhibitorsat MIC.

The bacterial growth curve measurement revealed how the bacterialpopulations were affected by JA-144, TH-4 and TH-8 at their MICs. The ODvalues provided quantitative information and thus narrowed down the MICsof JA-144, TH-4 and TH-8 to specific values. However, the OD method is arough estimate, and the bacterial growth is not observed with OD valueuntil the cell concentration reached approximately 10⁷ CFU/ml (Christinaet al., 2014). Thus, it could be concluded that even a small increaseobserved in the OD values in these experiments could mean thatsignificant changes in microbial populations occurred. Therefore,statistical analysis of these OD measurements could not objectivelyreflect differences in the microbial populations.

4.3. Antimicrobial Activities of JA-144, TH-4 and TH-8 with and withoutFilms against E. coli K12, L. innocua, C. jejuni and C. coli at eachMIC.

Typically, when an antimicrobial agent is incorporated into a film orbonded to the surface as a coating, it could lose some of its efficacy.If the functional groups of the antimicrobial agent bind too tightly tothe polymer, the releasing rate will be highly restricted when theycontact a contaminated surface (Han, 2000). Consequently, growth of themicroorganism will continue before the antimicrobial agent is released.On the other hand, if the releasing rate of antimicrobial agent isfaster than the growth rate of the target microorganism, theantimicrobial agent could be depleted and loses its efficacy quicklysince packaged food has an almost infinite volume compared to the amountof the antimicrobial agent (Raija, 2003). In addition, the processingmethod used in the manufacture of the film could act to interfere withthe antimicrobial function of the active agent (Wen-Xian et al., 2015).For polymeric films, this could occur during the synthesis process, ifthat is when the antimicrobial agent is incorporated or it could occurduring the extrusion process, when the resin is converted into a film.This extrusion process melts the polymeric resin into a flowing liquidbefore cooling it into the final desired shape as a film.

Reduction in the activities of the antimicrobial compounds, whenincorporated into the films, could be attributed to the nature of thefunctional groups and if they interact negatively with those of thepolymer. For example, the hydroxyl group in JA-144 was more likely tointeract with the tapioca starch granules and glycerol since they arehydrophilic. However, as for C. jejuni and C. coli, the losses inefficacies for JA-144 were only 0.25% and 2.03%. These weresignificantly lower than the reductions obtained when exposed to E. coliK12 or L. innocua. This could be attributed to the microaerobic natureof the Campylobacter species and the fact that this lead to their slowgrowth rate. Based on this premise, it can be assumed that the releaserate of JA-144 and Campylobacter's growth rate were perfectly matched.

5. Conclusion

In this study, the antimicrobial effect of the synthesized smallmolecules (JA-144, TH-4 and TH-8) against E. coli K12, L. innocua, C.jejuni and C. coli were evaluated qualitatively and quantitatively. Itcould be concluded that JA-144 was most effective in inhibiting thegrowth of the all the test organisms when compared with TH-4 and TH-8.TH-4 showed better antimicrobial activities than TH-8. The Campylobacterspecies appeared to be more sensitive to the small molecule compoundswhen compared with E. coli K12 or L. innocua. Although the antimicrobialefficacy of the small molecule compounds appeared to be reduced afterthey were incorporated into the edible films, for JA-144 and TH-4, thereductions were not significant when compared to direct exposure of themicroorganisms to the antimicrobial agents. Thus, it could be concludedthat JA-144 has great potential to be incorporated into edible films andcould be used to package and reduce bacterial growth on ready-to-eatfoods. However, additional researches on the release rate of thecompounds from the films are needed since this is indispensable tounderstanding of how the molecules will behave within the film.Additionally, the mechanical properties of the films should beinvestigated to ascertain if and how the functionality would be affectedby the addition of small molecule compounds.

EXAMPLE 3 Abstract

This study incorporated antimicrobial small molecule compounds (JA-144,TH-4, and TH-8) into tapioca films and investigated how they influencedthickness, moisture content, water activity, gas permeation, morphology,thermal and mechanical properties of the films. Results showed that,unlike TH-4 and TH-8, JA-144 caused significant (p<0.05) changes tothickness, moisture content, and water activity of the films whencompared with the control film. JA-144 caused a small but significant(p<0.05) increase in water vapor permeation (WVP) while TH-4 and TH-8caused a small lowering of the WVP. For the oxygen permeation, JA-144and TH-4 cause a small but significant increase but TH-8 had nosignificant effect. Compared to the melt temperature of the control(122.63° C.), JA-144 reduced it to 119.84° C. while TH-4 and TH-8increased it to 126.49° C. and 130.68° C., respectively. X-raydiffraction testing showed that JA-144 did not induce crystallinitychanges, but TH-4 and TH-8 appeared to increase crystallinity andsubsequent crosslinking effects. The Dynamic Mechanic Analysis testsshowed that JA-144 had no effect on the glass transition temperature(T_(g)) and storage modulus, while TH-4 and TH-8 that caused slightincreases in T_(g) and storage modulus of the films. The JA-144 filmshad higher moisture content and water activities while these were lowerin the TH-4 and TH-8 films. These results indicate that JA-144 causedthe films to be more flexible, higher in moisture and lower in gasbarrier properties. TH-4 and TH-8 did the opposite by making the filmsstiffer, lower in moisture and better barriers to gasses.

1. Introduction

Increasing demands for environmental-friendly packaging material havetriggered sustained researches in the development of starch-basedbio-composite films. Starch is one of the most preferred green packagingmaterials due to its rapid biodegradable nature, renewable sources andavailability at relatively low cost (Liu et al., 2011). Films made fromstarch could be used to cover food surfaces, separate incompatible zonesand ingredients in complex food mixes, form a barrier against oxygen,aroma, oil and moisture, and used to make pouches or wraps for foodpackaging. Besides, edible films can be used as carriers of functionalagents, such as antioxidants or antimicrobials, and can be used toimprove the appearance of selected foods (Kester and Fennema, 1986).

Tapioca is obtained from the cassava plant, which is a significant cropin South America, Africa, Latin America and Asia, and is an economicalsource of starch (FAO, 2004). Films made from tapioca starch exhibitappropriate physical characteristics, since they are odorless,tasteless, colorless, and good barriers to oxygen. However, tapiocastarch-based films could show brittleness with inadequate mechanicalproperties if not properly made. To overcome this weakness, plasticizersare generally required because it could reduce the intermolecular forcesand increase the mobility of polymer chains, therefore improving theflexibility and extensibility of the film. Glycerol is one example of aplasticizer used in filmmaking. It shows stability and compatibilitywith hydrophilic bio-polymeric materials used for packaging (FernandezCervera et al., 2004). However, high water solubility and poor watervapor barrier limit the application of hydrophilic materials such asstarch-based films. To solve these problems, the blending of starch withdifferent biopolymers or the addition of hydrophobic materials such asoils or waxes have been proposed (Xu et al., 2005; Garcia et al., 2000;Anker et al., 2001).

Antimicrobial(AM) agents have been found to be effective againstfood-borne pathogens. It is reported that the effectiveness of AMpackaging is greater than direct addition of AM agents into foods due totwo factors. One is the lower release rate of the AM agent from thematerial to the food, thus enabling functionality over a longer period.The other factor is inactivation concerns (such as neutralization,hydrolysis, or dilution) when the AMs are directly added into the food.Various types of AM agents have been incorporated into edible films.Examples of these include benzoic, sorbic, propionic, and lactic acids,nisin, and lysozyme, to retard surface growth of bacteria, yeasts, andmolds on a wide range of products, including meats and cheeses (Islam etal., 2002; Lahellec, et al., 1981; Lueck, E., 1976; Moir, et al., 1992;Reddy, et al., 1982; Sofos, et al., 1981). Small organic molecules havealways been of interest to chemists and biochemists due to theircapability of exerting powerful effects on the functions ofmacromolecules that comprise living systems (Marian et al., 1997). Asone of the most important therapeutic agents, small organic moleculeshave benefits such as improved stability over peptides in oraladministration, synthetic accessibility, and optimization of conveniencefor compound bioactivity when compared with macromolecules (Pathania etal., 2009). Synthesized small molecules are generally used to affect thegrowth of bacteria in two ways: by killing the bacteria, or inhibitingthe growth of the bacteria. However, the incorporation of additives intothe matrix of a polymer may alter its mechanical and barrier properties,which are two important factors known to affect the performance ofedible films.

The objectives of this study were to investigate how the addition ofsynthesized small molecules (JA-144, TH-4 and TH-8) could affect atapioca starch-based film's thickness, morphology, moisture content,oxygen and water vapor permeabilities, glass transition and meltingtemperatures and mechanical properties.

2. Materials and Methods 2.1. Materials

Tapioca starch powder was purchased from a local supermarket inColumbus, Ohio. It was irradiated at the Ohio State University NuclearReactor Laboratory in order to achieve sterilization. This was used asthe main ingredient in the films. Distilled water was used to make asuspension of the starch powder. Glycerol≥99.0% (Sigma-Aldrich®) wasobtained from Fisher Scientific (Fisher Scientific, Fair Lawn, N.J.) andused as a plasticizer. Acetic Acid (ACS Reagent≥99.7%), DimethylSulfoxide (ACS Reagent≥99.9%). The synthesized small molecules referredto as JA-144, TH-4 and TH-8 were obtained from Dr. James Fuch'slaboratory at the College of Pharmacy, OSU. The chemical structures ofthese molecules are shown in FIG. 1.

3.2. Method 2.2.1. Preparation of Small Molecule Compounds.

The small molecule solutions were prepared by dissolving 100 mg of eachcompound at room temperature in 1 ml DMSO in separate test tubes toreach a concentration of 100,000 ppm. A vortex mixer was used to ensurethat each compound was well dissolved into a homogenous solution. Thesmall molecule stock solutions were then stored at −20° C.

The 2,3,5-Triphenyltetrazolium Chloride (TTC) stock solution wasprepared by dissolving 20 mg in 1 ml distilled water at room temperatureto reach a concentration of 20,000 ppm. A vortex mixer was used toensure that it was well dissolved. The TTC stock solution was stored at−20° C. for future use.

The range of concentrations for the small molecules in the filmsdepended on previous cytotoxicity assay conducted by Dr. EsperanzaCarcache de Blanco's laboratory in the College of Pharmacy at OSU. Thistest determined the highest concentrations of each compound to causelysis of human colon cells. The results showed that the values forJA-144, TH-4 and TH-8 were 200 μM. When converted to ppm, they were 66.9ppm, 61.1 ppm, 60.4 ppm, respectively. The survival rates of colon cellsfor JA-144, TH-4 and TH-8 at 200 μM were 99%, 60%, and 79%,respectively. Since JA-144 showed less toxicity on human colon cells,the starting concentration for this study was 100 ppm; while for TH-4and TH-8, the starting concentration were 50 ppm, respectively.

2.2.2. Preparation of Tapioca Films.

The film forming solutions were prepared using blends of tapioca starchand JA-144, TH-4 and TH-8 dissolved in acetic acid with glycerol as aplasticizer. This began when aliquots of 100 mg of each small moleculecompound was first dissolved in 1 ml acetic acid. Tapioca starch (5%w/w) and acetic acid solutions (0.7% w/w) with the small molecules and1.8% w/w glycerol were dissolved into 100 ml distilled water as shown inTable 6.

TABLE 6 Composition of Tapioca Film on Wet and Dry Percent Weight Basis.Wet basis Dry basis Composition Weight % Composition Weight % Tapiocastarch  5% Tapioca starch 72.78% Glycerol 1.8% Glycerol 26.20% Aceticacid 0.7% Small molecules  1.02% Small molecules 0.07%  Distilled water93.23%  Total 100%  Total  100%

All dispersions were heated in a water bath (70° C.) for 15 min withstirring until completely gelatinized. An Ultrasonic Sonicator(Graymills Co., Chicago, Ill.) was used to remove air bubbles from thegelatinized solutions. The edible films were prepared by casting thesolutions (107 g) into 10-inch radius Teflon plates. These were ovendried at 45±2° C. for 12 hours, then the dried films peeled off from theplate surfaces. The final concentration of each small molecule compoundin the dry film was approximately 1% w/w. The film thicknesses weremeasured using a Magna-Mike 8500 Thickness Gage (Olympus, Japan), withresolution of 0.001 mm. A total of 5 measurements per film, at variouslocations were taken to determine the average thickness.

2.2.1.2 Film Moisture Content and Water Activity Testing.

The moisture content of the prepared films was determined by agravimetric method. To accomplish this, the samples were dried at 105±2°C. in a laboratory oven (UNE PA, Memmert, Germany) until constant weightwas achieved (Jiang et al., 2010). The tests were done by usingapproximately 1.0 g film samples that were previously conditioned for 24hours at 23° C. and 50% relative humidity. These were placed inpreviously dried and cooled glass petri dish and kept in the oven for 8hours. Weights of the samples were taken before and after drying using a5 decimal point XSE Analytical Balance (±0.01 mg) (Mettler Toledo Co.Toledo, Ohio). All tests were conducted in triplicates and the averagevalues were recorded.

As for water activity measurement, each sample (2.0 g) was placed intoweighting scale, and their initial water activity was determined using awater activity meter (AquaLab®, Decagon Devices Inc., Pullman, Wash.,USA).

2.2.1.3. Oxygen Permeation Testing.

Oxygen (O₂) permeability of the films was tested using an OX-TRAN® Model2/21 Series OTR instrument (Mocon Inc., Minneapoils, Minn.). The methodof oxygen permeability testing was done according to the ASTM D3985method with some modifications. The oxygen transmission rates of thefilm samples were measured at 23° C. and 0% relative humidity. AnAluminum mask manufactured by Modern Controls Inc. (Minneapolis, Minn.)was placed on each film to make a test area of 5 cm². Tests wereperformed after 12 hours conditioning. Nitrogen carrier gas was used topurge the chamber of the diffusion cell while oxygen gas flowed over oneside of the sample. The flow rate of the nitrogen carrier gas was 10cm³/min. Oxygen gas that permeated through the film into the nitrogencarrier gas was transported to the detector at the flow rate of 10cm³/min. An oxygen-sensitive coulometric sensor was used to measure thequantity of oxygen that permeated the material (ASTM, 2004). The resultswere obtained using a Model 34401A multimeter manufactured byHewlett-Packard Company (Loveland, Colo.). Frequent calibration of theinstrument was performed with a standard PET film sample obtained fromModern Control Inc. (Minneapolis, Minn.). Duplicate measurements on twopouches for each condition were obtained.

2.2.11 Water Vapor Permeation Testing.

The WVP tests were conducted using the ASTM E96 (1996) Method with somemodifications. Test cups with 50 cm² open area were filled with 10 ganhydrous calcium sulfate to produce a relative humidity of 0% insidethe cup. The film samples were placed on top of the cup and sealed withan O-ring. A high vacuum silicone sealant was applied between the O-ringand the film samples, between the sealing lip of the cup and the sample,before clamping them with 4 screws. The cups containing the desiccantswere weighed to give the initial weight and then placed in a humiditychamber at room temperature (23±2° C.) and relative humidity of 55±2%.At an hour intervals the cups were weighed until a steady state wasreached. The water vapor transmission rate (WVTR) through the film wasestimated from the linear portion of the plot of weight gained versustime and the slope divided by the film exposure area according toEquation 3.2. Three replicates per film were tested. The WVP of the filmwas calculated by multiplying the

WVTR by the film's thickness and dividing saturated by the vaporpressure difference across the film and surface area of the sampleexposed to the storage environment.

$\begin{matrix}\left( {{Equation}\mspace{14mu} 3.3} \right) & \; \\{{WVTR} = \frac{Q}{t}} & (3.2) \\{{WVPC} = {{WVTR} \times {\frac{T}{A \times \Delta \; P}.}}} & (3.3)\end{matrix}$

Where: A is the surface area (m²) of the sample exposed to moisture;

ΔP is the driving force, describing the humidity difference between twosides of film (Pa). In this test, Relative Humidity (RH) was determinedby measuring dry bulb and wet bulb temperature during the tests and RHvalue was obtained by using Psychometric Chart. The driving force ΔP wasbased on Saturation Pressure of Water Vapor, which was corresponding todry bulb temperature during the test. In this case, water vaporsaturation pressure was 7.30×10³ Pa according to Saturation Pressure &Temperature Chart.

-   -   T is thickness of the film (m);    -   Q is weight change of WVT cups (g)    -   t is period of time (day)

All tests were conducted in triplicate and the units for WVPC wereg×m⁻¹×s⁻¹×Pa⁻¹.

3.3.3 Thermal analysis By Differential Scanning Calorimetry (DSC)

A TA instrument (Q2000, USA) differential scanning calorimeter equippedwith a data collection station was used to scan the thermal transitionsof the tested films. The samples were weighed (ranging from 5 to 7 mg)using a 5 decimal point XSE Analytical Balance (±0.01 mg) (MettlerToledo Co. Toledo, Ohio) in aluminum pans followed by sealing withinverted lids, for optimum thermal conductivity. The reference was anempty aluminum pan sealed in the same manner. Both pans were thenequilibrated at 20° C. for 30 sec to stabilize the baseline followed byscanning until 200° C. at a heating rate of 10° C. min⁻¹. Thermogramswere recorded and analyzed by the TA Instrument software (UniversalAnalysis 2000, Version 4.1D). The Tm (Melting temperature) wasidentified as the inflexion point of the baseline. Three replicates perfilm were tested.

3.3.4 X-ray Diffraction Analysis

X-ray diffraction was measured using a Rigaku Miniflex 600diffractometer with vertical goniometer was used (Cu Kα radiationλ=1.542 Π). Operation was performed at 40 kV and 20 mA. All samples weremounted on a glass and were attached to the equipment holder and theX-ray intensities were recorded with a scintillation counter in ascattering angel (2θ) range of 3-50° with a scanning speed of 1°/min.Distances between the planes of the crystals d(Π) where calculated fromthe diffraction angels (°) obtained in the X-ray pattern, according toBragg's law:

nλ=2dsin(θ)   (3.4)

Where λ is the wavelength of the X-ray bean and n is the order ofreflection.

From the scattering spectrum, the effective percent crystallinity offilms was determined, according to Hermans and Weidinger (1961), as theratio of the integrated crystalline intensity to the total intensity.Crystalline area was evaluated on the basis of the area of the mainpeaks. Due to the complexity of the system, the calculatedcrystallinities are not taken as absolute value, but are rather used forcomparative purposes.

3.3.5 Mechanical testing by Dynamic Mechanical Analysis (DMA)

The mechanical properties of the films were determined using astress-controlled Dynamic-Mechanical Analyzer (DMA 2980, TA Instruments,Surrey, England) at a frequency of 1 Hz from −80 to 100° C. with aheating rate of 5° C. min⁻¹. Film samples (25 mm in length, 5 mm inwidth, and 6-8 mil in thickness) were equilibrated at room temperature(23±2° C.) and relative humidity of 55±2% for 48 hours prior to theanalysis. The storage (′E) and loss modulus (″E) as well as tan δ of thefilm samples were monitored as a function of temperature. Threereplicates per film were tested.

2.3. Statistical Analysis.

All data were analyzed using the analysis of variance (ANOVA), andTukey's multiple comparisons test was used to determine the significantdifferences between the means at a level of p<0.05. The statisticalanalysis was used to compare and determine the significant effect of theaddition of JA-144, TH-4, TH-8 on the thickness, moisture content, andthe mechanical and barrier properties of the tapioca films. SPSS Version10.0 software (SPSS Inc., Chicago, Ill.) was used for this purpose. Dataare presented as mean and standard deviation of duplicate or triplicateanalysis.

4. Results and Discussion 3.2 Thickness, Moisture Content and WaterActivity.

The thickness of the films was average from 5 readings taken from 5random places on the films. Results are shown in Table 7. The filmthicknesses ranged from 0.171±0.006mm for the TH-4 films to0.198±0.004mm for JA-144 films. The thickness of the films did notsignificantly (p>0.05) increased after the addition of TH-4 and TH-8.However, it significantly (p<0.05) increased after the addition ofJA-144 when compared with the control film.

TABLE 7 A Summary of Thicknesses, Moisture Contents and Water Activityof Tapioca films with JA-144, TH-4 or TH-8. Moisture Sample CompoundThickness¹ Standard Content² Standard Water ID conc. % (mm) Deviation(%) Deviation Activity Control 0 0.173^(a) 0.005 12.900^(a) 0.210 0.266JA-144 1 0.198^(b) 0.004 16.021^(b) 0.664 0.270 TH-4 1 0.170^(a) 0.00610.183^(c) 0.107 0.252 TH-8 1 0.172^(a) 0.005 9.824^(c) 0.100 0.259¹Each data point is expressed as the mean of five measurements ²Eachdata point is expressed as the mean of three measurements ^(a-c)In agiven column, values with same letters are not significantly different(p > 0.05), while different letters are significantly different (p <0.05).

The moisture content and water activity of the Tapioca films with andwithout JA-144, TH-4, TH-8 (FIG. 25) were measured and are also shown inTable 7, and FIG. 17. From the results obtained, the JA-144 film had thehighest moisture content value and water activity of 16.021±0.664%,0.270.

3.4.2 Water Vapor Permeability.

The water vapor permeabilities of the films are shown in Table 8 andFIG. 26. As can be seen from the results, the WVP of the tapioca filmwithout compound was 6.546×10⁻¹¹±9.688×10⁻¹³ g×m⁻¹×s⁻¹×Pa⁻¹. The JA-144,TH-4, and TH-8 films produced WVP values of 8.183×10⁻¹¹±2.752×10⁻¹³,5.186×10⁻¹¹±1.386×10⁻¹², 5.389×10⁻¹¹±4.636×10⁻¹³ g×m⁻¹×s⁻¹×Pa⁻¹,respectively. The WVP of the films was significantly decreased (p<0.05)after the addition of TH-4 and TH-8. However, it was significantlyincrease (p<0.05) after the addition of JA-144 compared with the controlgroup.

TABLE 8 The summary of WVP calculations for Tapioca Films with JA-144,TH-4 or TH-8 Sample OTR (cc × Permeant OPC¹ (cc × m⁻¹ × Standard ID m⁻²× s⁻¹) Conc. (%) s⁻¹ × Pa⁻¹) Deviation Control 1.519 × 10⁻⁵ 21 8.564 ×10^(−14a) 3.489 × 10⁻¹⁵ JA-144 1.470 × 10⁻⁵ 21 1.084 × 10^(−13b) 2.708 ×10⁻¹⁵ TH-4 1.241 × 10⁻⁵ 21 7.406 × 10^(−14c) 4.206 × 10⁻¹⁵ TH-8 1.195 ×10⁻⁵ 21 8.310 × 10^(−14a) 1.247 × 10⁻¹⁵

3.3.6 Oxygen Permeability Coefficient.

The Oxygen Permeability Coefficients (OPC) of the films are shown inTable 9 and FIG. 27. As can be seen from the results, the OPC of thetapioca film without compound was 8.564×10⁻¹⁴±3.469×10⁻¹⁵cc×m⁻¹×s⁻¹×Pa⁻¹. The JA-144, TH-4, and TH-8 films produced OPC values of1.084×10⁻¹³±2.708×10⁻¹⁵, 7.406×10⁻¹⁴±4.206×10⁻¹⁵,8.310×10⁻¹⁴±1.247×10⁻¹⁵cc×m⁻¹×s⁻¹×Pa⁻¹, respectively. The OPC of thefilms did not significantly decreased (p<0.05) after the addition ofTH-8. However, it was significantly increased (p<0.05) after theaddition of JA-144 or decreased on the addition of TH-4 and TH-8 whencompared with control group.

TABLE 9 The Summary of OPC calculations for Tapioca Films with JA-144,TH-4 or TH-8. Relative Sample WVTR Humidity¹ WVP¹ (g × m⁻¹ × Standard ID(g/s) (%) ΔP (Pa) s⁻¹ × Pa⁻¹) Deviation JA-144 8.344 × 10⁻⁶ 56.9 4.15 ×10³ 8.183 × 10^(−11a) 2.752 × 10⁻¹³ TH-4 5.962 × 10⁻⁶ 56.9 4.15 × 10³5.186 × 10^(−11b) 1.386 × 10⁻¹² TH-8 6.323 × 10⁻⁶ 56.9 4.15 × 10³ 5.389× 10^(−11b) 4.636 × 10⁻¹³ Control 7.708 × 10⁻⁶ 56.9 4.15 × 10³ 6.546 ×10^(−11c) 9.688 × 10⁻¹³ ¹Each data was expressed as the mean of threemeasurements. ^(a-c)In a given column, values with same letters are notsignificantly different (p > 0.05), while different letters aresignificantly different (p < 0.05).

3.3.7 Thermal Analysis by Differential Scanning Calorimetry

The differential Scanning calorimetric curves in FIG. 28 display thethermally induced endothermic transitions for JA-144, TH-4 and TH-8tapioca films from 20° C. to 200° C. As shown in FIG. 28, the meltingtemperature (T_(m)) of the control, JA-144, TH-4 and TH-8 films were122.63° C., 119.84° C., 130.68° C., and 126.49° C., respectively. Boththe control and JA-144 films exhibited a single endothermic peak, whichindicated homogeneity of the films. This is an indication that JA-144blended well within the molecular structure of the starch. For the TH-4or TH-8 films, an extra small peak was observed at 87.14° C. or 90.95°C., respectively, suggested that TH-4 or TH-8 was not completelyincorporated into the molecular structure of the starch film. Theresults also reflected that the addition of JA-144, TH-4, and TH-8caused a shift in the T_(m), indicated the film's crystallinity wasaffected by the compounds. In addition, a broadening of the peaks whenthe compounds were added to the starch is an indication of increasedvariability of the crystals within the molecular structure of thestarch.

3.3.8 X-ray Diffraction Pattern

The X-ray patterns of Tapioca film only, or with 1% w/w JA-144, TH-4, orTH-8 films were presented in Figure 296. The main peak positions of eachsample were summarized in Table 10.

TABLE 10 The position of main X-ray diffraction peaks of Tapioca Filmswith JA-144, TH-4 or TH-8 Sample ID Peak Position) Control 3.80 7.6011.39 38.70 7.20 21.20 28.54 19.43 JA-144 3.80 7.60 11.39 38.70 21.2028.54 19.43 TH-4 3.80 11.39 38.70 TH-8 3.80 7.60 11.39 38.70 21.20 28.54

As shown in FIG. 29, all the samples showed different extent ofcrystallinity. The Tapioca film matrix was mostly amorphous in naturewith one diffuse peak located at 21.20° and other small crystallinefraction imbedded in the amorphous matrix as indicated by small hump atvarious locations. By adding JA-144 into the film, it was showed thatthe intensity of main diffuse peak at 21.20° increased, and one of thesmall peaks (7.20° disappeared. By adding TH-4 into the film, the X-raypattern was apparently changed. It was showed that a strong peakappeared at 3.80° and most of the small peaks disappeared. The samepattern was observed on TH-8 film. It was showed that a strong peakappeared at 7.60° and some of the small peaks disappeared, respectively.

3.3.9 Mechanical testing by Dynamic Mechanical Analysis (DMA)

In Dynamic Mechanical Analysis (DMA) experiments, information on thestorage modulus, loss modulus and tan δ of the tapioca films withJA-144, TH-4, and TH-8 were obtained. The data were used to determineglass transition temperature T_(g) as well as the stress-strain curve asa function of time. By definition, temperature corresponding to a sharpdecrease in storage modulus, or a maximum value of loss modulus and tanδ during a temperature sweep is the glass transition temperature(Sperling, 2001). Storage modulus provides important information offilm's ability to store energy in response to an applied force at giventemperatures. It is also called elastic modulus and relates to theinherent stiffness of the sample. FIG. 30 shows the effects of JA-144,TH-4, TH-8 addition on the storage modulus over the entire temperaturerange of the DMA. The data show that the addition of JA-144, TH-4, andTH-8 had significant effects (p<0.05) on the modulus of the test films.Particularly, the addition of JA-144 decreased the storage modulus attemperature above −57° C., while the addition of TH-4 or TH-8significantly increased storage modulus at temperatures above −14° C.,as shown in FIG. 30.

The results shown in FIGS. 31 and 32 were used to determine the effectof JA-144, TH-4, or TH-8 on the loss modulus and tan δ of the films.From this information the T_(g) for the films were determined, which wasestimated to be −67.249° C. and −67.249° C., respectively. As for TH-4and TH-8 film, T_(g) values were the highest, which were −66.508° C. and−66.508° C., respectively.

3.4 Discussion

3.5.1 Thickness, Moisture Content, Water Activity, and barrierproperties (OPC, WVP)

The results showed that thicknesses of the films did not significantlyincreased after the addition of TH-4 and TH-8. However, it wassignificantly increased after the addition of JA-144 compared withcontrol group. The thickness increase caused by JA-144 was possibly dueto the hydroxyl groups in the compound and its hydrogen bonding to thepolymeric chains (Lagos et al., 2015). The existence of hydroxyl groupin JA-144 also played a key role in film's moisture content and wateractivity due to its hygroscopic character. This interaction with thefilm's matrix increased the spaces between the chains, thus facilitatingwater migration into the film, and consequently, increasing its moisturecontent and water activity (da Matta, et al., 2011). These structuralchanges within the JA-144 films further affected their barrierproperties, which are shown in the WVP and OPC tests. As shown in Table8 and Table 9, the WVP and OPC of JA-144 are 8.183×10⁻¹¹ g×m⁻¹×s⁻¹×Pa⁻¹,1.084×10⁻¹³ cc×m⁻¹×s⁻¹×Pa⁻¹, respectively, which are 25.00% and 26.58%higher than the control film. (Chien-Hsien, et al., 2007).

As for TH-4 and TH-8 films, the results indicate that there was nosignificant change in thickness, and they had lower moisture content andwater activity, but good water and oxygen barrier properties. Thesecould be attributed to the sulfonamide functional group in TH-4 andTH-8. The sulfonamide functional group is rigid, hydrophobic, andtypically has a tendency toward crystallization (Seong et al., 2001).These characters of sulfonamide functional group in TH-4 and TH-8 couldprobably enhance the crosslinking effect within the film matrix, andconsequently lead to less moisture content, less water activity, butgood oxygen and water barrier properties.

3.5.2 Mechanical and Thermal Properties

As discussed previously, JA-144 film showed hydrophilic characteristics,while TH-4 and TH-8 films were hydrophobic, rigid, and had a tendencytoward crystallization due to the sulfonamide functional group.Therefore, it is expected that JA-144 film should have lower T_(g),lower T_(m), and less storage modulus due to its low crystallinity,higher amorphous, and high flexibility characteristics compared to thecontrol film. As for TH-4 and TH-8 films, it is expected that theyshould show a higher T_(g), higher T_(m), and more storage modulus dueto the presence of halogens in their structure. These have a tendency toincrease crosslinking and subsequently increase the crystallinity of thepolymer (Jianwei et al., 2005).

These expectations were proved by the DSC, X-ray diffraction and DMAtests. The Tm of JA-144 was 119.84° C., which is 2.28% lower than thatof the control; while for TH-4 and TH-8 films, they are 3.15% and 6.56%higher than the control, respectively. The storage modulus reflected thestiffness of film, showed a value −57° C. for JA-144 film, and this waslower than the control film. For the TH-4 and TH-8 films, higher storagemodulus values were expected and the results show that these values weresignificantly higher than the control film (above −11° C.), as shown inFIG. 30.

X-ray diffraction tests further detect these minor changes. As shown inFIG. 3.5, the addition of JA-144 resulted in a slightly increase ofcrystallinity. This could be due to the intra-molecular interactions ofhydroxyl groups between amylose and JA-144 that led to the formation ofintramolecular hydrogen bonding. No extra peaks were observed indicatedthat JA-144 was well blended. However, the addition of TH-4 and TH-8resulted in a dramatic increase of crystallinity. X-ray patterns forthese two were significantly changed, which could be ascribed to twopossibilities. First, it is possible that crosslinking effect betweenTH-4/TH-8 and starch amylose were formed when adding TH-4/TH-8 into filmforming solution. Crosslinking restricts molecular mobility, tying thepolymer backbones together thus results in crystallinity increase(Shulamit et al., 2008). Second, it is possible that TH-4/TH-8 moleculesare not well blended into the film. Previous research demonstrated thatsulfonamide group is rigid, hydrophobic, and typically has a tendencytoward crystallization (Seong et al., 2001). Since the film formingsolution is hydrophilic, it is possible that these TH-4/TH-8 moleculeswere self-assembled or aggregated during the blending process (Kazunariet al., 1992). As a result, a high intensity peak shown in X-raydiffraction was observed.

An ideal edible film with incorporated compounds for antimicrobialapplication should have less moisture content, good barrier properties,and good mechanical properties (Bhanu et al., 2015). Based on thesecriteria, JA-144 film showed high moisture content, high OPC and WVP,which means low barrier properties, but good mechanical properties dueto the plasticizing and co-plasticizing effect of hydroxyl group withwater molecules. This means that the JA-144 films will dissolve fasterin the mouth and needs less chewing since it showed a lower mechanicalstrength and higher moisture holding capacity. A possible mechanism ofJA-144 binding to amylose or glycerol is shown in FIG. 33. As for TH-4and TH-8 films, they showed less moisture content, low OPC and WVP,which means good barrier properties, but low mechanical strength due tothe crosslinking effect of sulfonamide group. This means the TH-4 andTH-8 films show better characteristics for applications such aspackaging and wrapping, which require more mechanical strength and lessgas permeability. Also, these compounds were not completely incorporatedinto the film as both DSC curves showed an extra peak at 87.14° C. and90.95° C. Consequently, future work should be focused on how to optimizethe structure of these molecules, or to introduce another chelatingagent to offset drawbacks of each compound. By doing so, it is to beexpected that these compounds could be better incorporated into thefilm.

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What is claimed is:
 1. A composition of matter comprising anantimicrobial compound, said composition comprising a compound selectedfrom JA-144, TH-04 or TH-08 or combination thereof, said compound havingthe following formula:

and derivatives, modifications, or pharmaceutically acceptable saltsthereof
 2. The composition of matter of claim 1 further comprising acarrier.
 3. The composition of matter of claim 2, wherein said carriercomprises a pharmaceutically acceptable excipient.
 4. The composition ofmatter of claim 3, comprising a composition for topical administration.5. The composition of matter of claim 3, comprising a composition forsystemic administration.
 6. The composition of matter of claim 2,wherein said carrier comprises a film.
 7. The composition of matter ofclaim 6, wherein said compound comprises JA-144.
 8. The composition ofmatter of claim 6, wherein said film comprises an edible film.
 9. thecomposition of matter of claim 8, wherein said compound comprisesJA-144.
 10. The composition of matter of claim 6, wherein said filmcomprises a package.
 11. The composition of matter of claim 10, whereinsaid compound comprises TH-4 or TH-8 or combination thereof.
 12. Amethod of treating a subject in need of treatment for a bacterialinfection, said method comprising administering to said subject aneffective amount of a composition comprising a compound of JA-144, TH-04or TH-08 or combination thereof, having the following formula:


13. The method of claim 12, said composition further comprising acarrier.
 14. The method of claim 12, wherein said carrier comprises apharmaceutically acceptable excipient.
 15. The method of claim 12,wherein said subject is a mammanl.
 16. The method of claim 12, whereinsaid subject is poultry and said bacterial infection comprises aCampylobacteria infection.
 17. A method of inhibiting bacterial growthon a surface, the method comprising contacting said surface with acomposition of matter comprising an effective amount of a compound ofJA-144, TH-04 or TH-08 or combination thereof, having the followingformula:

and derivatives, modifications, or pharmaceutically acceptable saltsthereof.
 18. The method of claim 17, wherein said compositions comprisesa carrier.
 19. The method of claim 18, wherein said carrier comprises afilm.
 20. The method of claim 18, wherein said carrier comprises anedible film.